Announcing the call for the Special Issue on the 20th International Congress of the International Union of Pure and Applied Biophysics (IUPAB)—virtual meeting,October 2021

This Commentary describes a call for submissions for the upcoming Special Issue focused on the science presented at the 20^th IUPAB Congress to be held in conjunction with the 45^th Annual Meeting of the Brazilian Biophysical Society and the 49^th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology.

Due to the COVID-19 pandemic, the 20^th International IUPAB Congress will take place as a virtual meeting this year from October 4 to 8, 2021. This triennial IUPAB Congress will be held in loose conjunction with the 45^th Annual Meeting of the Brazilian Biophysical Society and the 49^th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology. To act as a complement to this virtual meeting, the Biophysical Reviews journal will base a Special Issue on the scientific topics of the meeting contributors selected from the range of invited speakers and poster presenters. This Special Issue will also work to highlight the host country’s (Brazil) National Biophysical Society. Finally, this Special Issue will also serve to publish the meeting abstracts in supplemental form.Review articles from IUPAB Congress speakers and poster presenters to the IUPAB Congress and associated conferences (the 45^th Annual Meeting of the Brazilian Biophysical Society and the 49^th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology) are solicited. Similar to the SI based on the 19^th IUPAB Congress held in Edinburgh summarizing Commentaries from session chairs are also requested (Hall and dos Remedios 2017). The Special Issue for the 20^th IUPAB International Congress will be prepared and edited by the current authors (Rosangela Itri, Mauricio Baptista, Richard Garratt, and Antonio Jose Costa-Filho).     

Biophysical Reviews— 2021, the year that was

The current issue (volume 13 issue 6, 2021) is a Special Issue jointly dedicated to scientific content presented at the 20th triennial IUPAB Congress that was held in conjunction with both the 45th Annual Meeting of the Brazilian Biophysical Society (Sociedade Brasileira de Biofísica - SBBf) and the 50th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (Sociedade Brasileira de Bioquímica e Biologia Molecular – SBBq). In addition to describing the scientific and nonscientific content arising from the meeting this sub-editorial also provides a look back at some of the high points for Biophysical Reviews in the year 2021 before going on to describe a number of matters of interest to readers of the journal in relation to the coming year of 2022.

This Editorial marks the last issue for the journal to be published in 2021 – a year that has been characterized by a mixture of hardship, frustration, and of late, (possibly) a slowly developing cautious optimism in relation to the COVID-19 pandemic. Over the last 2 years, the journal has had to rapidly adapt to suddenly altered plans of contributors, as the publication of scientific reviews and organization of conference-based special issues has necessarily taken a back seat to the realities of altered work practices and, in some cases, changed life and career plans. One such major change was directly concerned with the subject of this special issue (SI) on the scientific content associated with the 20th Congress of the IUPAB (International Union for Pure and Applied Biophysics) conducted in concert with the 45th Annual Meeting of the Brazilian Biophysical Society (SBBf) and the 50th Annual Meeting of the Brazilian Biochemical and Molecular Biology Society (SBBq) (Itri et al. 2021). After discussing a few notable features of the SI, this editorial will introduce important developments occurring with the journal that relate to new feature commentaries and Institutional access arrangements. This Editorial will then close with a look back at some of the standout articles of 2021.     

A New Extracellular β-Galactosidase Producing Kluyveromyces sp. PCH397 from Yak Milk and Its Applications for Lactose Hydrolysis and Prebiotics Synthesis

β-Galactosidase is a crucial glycoside hydrolase enzyme with potential applications in the dairy, food, and pharmaceutical industries. The enzyme is produced in the intracellular environment by bacteria and yeast. The present study reports yeast Kluyveromyces sp. PCH397 isolated from yak milk, which has displayed extracellular β-galactosidase activity in cell-free supernatant through the growth phase. To investigate further, cell counting and methylene blue staining of culture collected at different growth stages were performed and suggested for possible autolysis or cell lysis, thereby releasing enzymes into the extracellular medium. The maximum enzyme production (9.94 ± 2.53U/ml) was achieved at 37 °C in a modified deMan, Rogosa, and Sharpe (MRS) medium supplemented with lactose (1.5%) as a carbon source. The enzyme showed activity at a wide temperature range (4–50 °C), maximum at 50 °C in neutral pH (7.0). In addition to the hydrolysis of lactose (5.0%), crude β-galactosidase also synthesized vital prebiotics (i.e., lactulose and galacto-oligosaccharides (GOS)). Additionally, β-fructofuranosidase (FFase) activity in the culture supernatant ensued the synthesis of a significant prebiotic, fructo-oligosaccharides (FOS). Hence, the unique features such as extracellular enzymes production, efficient lactose hydrolysis, and broad temperature functionality by yeast isolate PCH397 are of industrial relevance. In conclusion, the present study unrevealed for the first time, extracellular production of β-galactosidase from a new yeast source and its applications in milk lactose hydrolysis and synthesis of valuable prebiotics of industrial importance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00955-1.Keyword: β-Galactosidase, Lactulose, Galacto-oligosaccharides, Fructo-oligosaccharides, Milk-microbes

β-Galactosidase (EC 3.2.1.23) hydrolyzes the glycosidic bond in β-galactosides and finds applications in the food industry [1, 2]. The trans-glycosylation property of β-galactosidase (β-gal) is widely used to produce various galactosylated products and prebiotics such as GOS and lactulose [3–7]. The β-gal enzyme is produced intracellularly by many bacteria and yeast, a major constraint for industrial production [1, 8]. Therefore, extracellular β-gal producing bacteria/yeast are of huge relevance. Hence, the present work revealed an efficient extracellular β-gal producing microbe from dairy products of the Indian Himalaya and evaluated its applications in lactose hydrolysis and prebiotics’ synthesis.In this study, twenty milk and four curd samples were collected from the Lahaul and Pangi valleys of Himachal Pradesh, India. The samples were plated on MRS and Elliker agar medium (Himedia, India) for 2–7 days at 28 °C and 37 °C until visible microbial growth. Morphologically distinct isolates were screened for β-gal activity using X-Gal and IPTG plate assay [6, 9]. The positive isolates were screened for β-gal production in liquid MRS medium. The β-gal activity was expressed as U/mg dcw (dry cell weight) for whole cells and U/ml for cell-free supernatant [10, 11]. Yeast isolate PCH397 showing the highest and extracellular enzymatic activity was selected. The culture and reaction conditions for maximum β-gal activity were optimized. FFase activity of whole cells and cell-free supernatant was estimated as described by Lincoln and More [12].The cell-free supernatant (β-gal) was employed for applications in lactose hydrolysis and prebiotic synthesis. The enzyme was incubated with lactose solution (5%, w/v) at 37 °C for lactose hydrolysis followed by thin layer chromatography (TLC) [13] analysis and quantification using the ImageJ program (http://rsbweb.nih.gov/ij/). Further, the cell-free supernatant was incubated with milk at 4 °C for milk lactose hydrolysis. Samples were withdrawn at different time intervals and analyzed for residual lactose concentration using ultra-high performance liquid chromatography-quadrupole-time of flight-ion mobility mass spectrometry (UHPLC-Q-TOF-IMS) [14]. Prebiotic production was carried out by mixing an equal volume of the enzyme with a sugar solution i.e., lactose (40%, w/v) for GOS, and lactose (20%, w/v) + fructose (20%, w/v) for lactulose and FOS production, respectively at 50 °C for 24 h [6]. Samples were analyzed by TLC for GOS, UHPLC-Q-TOF-IMS for FOS and lactulose synthesis.The study resulted in the isolation of 203 morphologically distinct microbes, 62 of which were tested positive for β-gal. Based on quantitative screening, eight isolates showing maximum β-gal activity were selected and examined for the intracellular and extracellular enzymatic activities (Table S1). Yeast isolate PCH397 exhibited maximum extracellular β-gal activity (9.94 ± 2.53 U/ml) along with FFase activity (0.59 ± 0.155) after 48 h of incubation. Isolate PCH397 was identified as Kluyveromyces marxianus by its morphological and molecular characterization (Fig. S1). Phylogenetic tree based on ITS DNA sequence showed similarity (99.63%) with Kluyveromyces marxianus CBS712. To the best of our knowledge, the genus Kluyveromyces has not been reported earlier for extracellular β-gal production. In the past, efforts were made to produce β-gal extracellularly through permeabilization or incorporation of signal peptide to β-gal gene in a fusion construct [15, 16]. The isolate PCH397 was selected due to its generally regarded as safe (GRAS) status and the novel feature of extracellular enzyme production.Highest β-gal activity in the extracellular environment was observed when PCH397 was grown in MRS medium supplemented with 1.5% (w/v) lactose as a substrate and incubated at 37 °C for 48 h (Fig. S2). PCH397 produced extracellular β-gal at lower lactose concentration (1.5%) as compared to various Kluyveromyces spp. [15] where 3% lactose has been used in the growth medium for intracellular β-gal production. Further, whether the extracellular enzyme activity is due to the secretion or cell lysis, the CFU count and cell viability were checked by the methylene blue test. The decreased cell count in the late stationary phase for live cells (Fig. S3) and increased number of methylene blue stained cells indicated cell death (Fig S4). These results suggested that cell lysis in the late stationary phase leads to the secretion of enzymes in extracellular medium. The extracellular production of enzyme would lead to a lower production costs of the enzyme.Cell-free supernatant showed the highest β-gal activity at pH 7.0 in 10 mM sodium phosphate buffer at 50 °C in 5 min (Fig S2). The β-gal enzyme from the current finding holds promise in the sweet whey and milk lactose hydrolysis [1] due to its neutral pH optima. Also, β-gal, which is functional at high temperatures, is used in the synthesis of oligosaccharides [1, 3]. High temperature increases the reaction rate as well as lactose solubility, thus, facilitating transgalactosylation reactions [17]. The β-gal activity (9 U/ml) in cell-free supernatant of PCH397 completely hydrolyzed 5.0% of lactose within 8 h at 37 °C (Fig. 1a, S5a). In a recent study, 5.0% lactose was also hydrolyzed by purified β-gal (5 U/ml) of Paenibacillus barengoltzii CAU904 within 8 h at 40 °C [13]. Under refrigerated conditions (4 °C), the cell free supernatant hydrolyzed ~ 50% milk lactose within 36 h and ~ 80% in 72 h (Fig. 1b, S5b). Since β-gal of PCH397 is active at 4 °C, the enzyme could be utilized to hydrolyze lactose in dairy products under refrigerated conditions. Lactose-free milk products or low-lactose milk products are important dietary constituents for lactose intolerant individuals and deliver essential nutrients to combat nutritional deficiencies [18]. Even with commercially purified enzymes, 100% milk-lactose hydrolysis could not be achieved at a low temperature [19]. However, the crude enzyme from the present investigation can efficiently hydrolyze milk lactose at ambient and refrigerated conditions, reducing the cost associated with enzyme purification. Additionally, the source of enzyme is Kluyveromyces sp. which has GRAS status, therefore, can be used in food applications.Open in a separate windowFig. 1Lactose hydrolysis by crude β-gal of PCH397. a Relative quantification of the hydrolysed products from lactose (5%, w/v) at 37 °C for 24 h. b Relative decrease in lactose concentration (%) at refrigerated conditions obtained by UHPLC-QTOF-IMSFurther, the enzyme was evaluated for its ability to catalyze transgalactosylation reactions at 50 °C. The crude enzyme was incubated with different substrate mixture viz. lactose and fructose. After 8 h of incubation, 50% of lactose was hydrolyzed into glucose, galactose, and GOS (Fig. S6a). Maximum GOS production was achieved after 12 h (Fig. 2a). The purified β-gal from Paenibacillus barengoltzii synthesized GOS from 350 g/L of lactose within 4 h [13]. Though GOS synthesis was faster in comparison to the current study, it is to be noted that we used a crude enzyme mixture instead of a purified enzyme. The crude enzyme has also shown FFase activity (Table S1), and was used for the synthesis of FOS from lactose and fructose mixture. UHPLC-Q-TOF-IMS analysis confirmed the formation of FOS (Fig. 2b). Multiple peaks were observed in the sample containing lactulose, one of which was identical with the peak of lactulose standard (Fig. 2c) as confirmed by HPAEC-PAD (Fig. S6b). The lactulose formation was maximum at 20 h of incubation (Fig. S6c).Open in a separate windowFig. 2Hydrolysis and transgalactosylation of lactose by crude enzyme from PCH397 having β-gal and FFase activity. a Relative quantification of the hydrolyzed and transgalactosylated products. UHPLC-QTOF-IMS detection of prebiotics b FOS and c lactulose with their respective standardIt is the first report of simultaneous co-synthesis of multiple prebiotics i.e., GOS, FOS, and lactulose using a yeast strain. Similar reports for GOS and FOS synthesis have been attempted by enzymatic means from fungal sources in the past [6]. The synthesis of multiple prebiotics is very advantageous. Numerous studies have shown that blended consumption of multiple prebiotics including GOS and FOS has many health benefits [20–24]. The combination of GOS, FOS, and lactulose can be of considerable importance for their prebiotic applications. In conclusion, our findings revealed a yeast source for the cost-effective production of β-galactosidase and a strategy for co-synthesis of valuable prebiotics, which is not reported in the past. The utilization of a yeast source with GRAS status for lactose hydrolysis and co-synthesis of prebiotics promises various health benefits and commercial relevance.     

Safety and feasibility of umbilical cord mesenchymal stem cells in treatment-refractory systemic lupus erythematosus nephritis: time for a double-blind placebo-controlled trial to determine efficacy

As translational clinical researchers familiar with the risk-benefit of hematopoietic stem cell transplantation in autoimmune diseases, we are intrigued by the recent report of umbilical cord mesenchymal stem cell (UC-MSC) transplantation in treatment-refractory systemic lupus erythematosus nephritis by Wang and colleagues. They report the results of an open-label single-arm multicenter phase I/II study. This stimulated us to examine whether collective data from this group provide sufficient evidence for the feasibility, safety, dose rationale, and potential efficacy of UC-MSCs to conduct a randomized controlled trial in such patients. Results, though confounded by variable baseline prednisone and immuno-suppressive treatment, appear to indicate near-term response rates of approximately 50%, which are comparable to those seen with hematopoietic stem cell transplantation but with less morbidity and mortality.Wang and colleagues have been in the forefront of evaluating the potential for mesenchymal stem cells (MSCs) to treat systemic lupus erythematosus (SLE), based on studies in murine autoimmune disease models, demonstrating immunomodulatory properties of MSCs [1]. We have reviewed the additional reports published in peer-reviewed journals from this group [2–5], together with two protocols available on ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT00698191","term_id":"NCT00698191"}}NCT00698191 and {"type":"clinical-trial","attrs":{"text":"NCT01741857","term_id":"NCT01741857"}}NCT01741857) (Table 

Cancer resistance,high molecular weight hyaluronic acid,and longevity

Longevity varies greatly among mammals. The naked mole rat is among the longest-lived rodents, having an average lifespan of 32 years, compared to the similarly-sized house mouse with lifespan of 4 years. The rate of cancer also varies widely among mammals and interestingly, the naked mole rat is essentially cancer-free (Gorbunova et al., Nat Rev Genet 15(531):540, 2014). A series of elegant studies (Tian et al. Nature 499:346–349, 2013) has revealed that this cancer resistance derives from the abundant production of high molecular weight hyaluronic acid. Remarkably, high molecular weight hyaluronic acid, which accumulates within the extracellular matrix, stimulates an intracellular pathway that induces expression of p16^ink4a and suppresses oncogenic transformation.     

The NOTCH signaling pathway in normal and malignant blood cell production

The NOTCH pathway is an evolutionarily conserved signalling network, which is fundamental in regulating developmental processes in invertebrates and vertebrates (Gazave et al. in BMC Evol Biol 9:249, 2009). It regulates self-renewal (Butler et al. in Cell Stem Cell 6:251–264, 2010), differentiation (Auderset et al. in Curr Top Microbiol Immunol 360:115–134, 2012), proliferation (VanDussen et al. in Development 139:488–497, 2012) and apoptosis (Cao et al. in APMIS 120:441–450, 2012) of diverse cell types at various stages of their development. NOTCH signalling governs cell-cell interactions and the outcome of such responses is highly context specific. This makes it impossible to generalize about NOTCH functions as it stimulates survival and differentiation of certain cell types, whereas inhibiting these processes in others (Meier-Stiegen et al. in PLoS One 5:e11481, 2010). NOTCH was first identified in 1914 in Drosophila and was named after the indentations (notches) present in the wings of the mutant flies (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010). Homologs of NOTCH in vertebrates were initially identified in Xenopus (Coffman et al. in Science 249:1438–1441, 1990) and in humans NOTCH was first identified in T-Acute Lymphoblastic Leukaemia (T-ALL) (Ellisen et al. in Cell 66:649–61, 1991). NOTCH signalling is integral in neurogenesis (Mead and Yutzey in Dev Dyn 241:376–389, 2012), myogenesis (Schuster-Gossler et al. in Proc Natl Acad Sci U S A 104:537–542, 2007), haematopoiesis (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010), oogenesis (Xu and Gridley in Genet Res Int 2012:648207, 2012), differentiation of intestinal cells (Okamoto et al. in Am J Physiol Gastrointest Liver Physiol 296:G23–35, 2009) and pancreatic cells (Apelqvist et al. in Nature 400:877–881, 1999). The current review will focus on NOTCH signalling in normal and malignant blood cell production or haematopoiesis.     

Peroxisome-driven ether-linked phospholipids biosynthesis is essential for ferroptosis

It is well established that ferroptosis is primarily induced by peroxidation of long-chain poly-unsaturated fatty acid (PUFA) through nonenzymatic oxidation by free radicals or enzymatic stimulation of lipoxygenase. Although there is emerging evidence that long-chain saturated fatty acid (SFA) might be implicated in ferroptosis, it remains unclear whether and how SFA participates in the process of ferroptosis. Using endogenous metabolites and genome-wide CRISPR screening, we have identified FAR1 as a critical factor for SFA-mediated ferroptosis. FAR1 catalyzes the reduction of C16 or C18 saturated fatty acid to fatty alcohol, which is required for the synthesis of alkyl-ether lipids and plasmalogens. Inactivation of FAR1 diminishes SFA-dependent ferroptosis. Furthermore, FAR1-mediated ferroptosis is dependent on peroxisome-driven ether phospholipid biosynthesis. Strikingly, TMEM189, a newly identified gene which introduces vinyl-ether double bond into alkyl-ether lipids to generate plasmalogens abrogates FAR1-alkyl-ether lipids axis induced ferroptosis. Our study reveals a new FAR1-ether lipids-TMEM189 axis dependent ferroptosis pathway and suggests TMEM189 as a promising druggable target for anticancer therapy.Subject terms: Phospholipids, Cancer metabolism

Ether phospholipids represent an important group of phospholipids containing a glycerol backbone with an alkyl or a vinyl bond connecting a fatty alcohol at sn-1 position, usually polyunsaturated fatty acid (PUFA) including docosahexaenoic acid and arachidonic acid at sn-2. Ether phospholipids are initially synthesized in peroxisomes and processed in the endoplasmic reticulum (ER) [1–3]. Plasmalogens are the most abundant form of ether phospholipids which have a vinyl ether bond, enriched in the brain and heart tissues [1–3]. The plasmalogens have been found as endogenous antioxidants with vinyl ether bond susceptible to cleavage by reactive oxygen species (ROS). The deficiency of plasmalogens correlates with various human disorders, including Alzheimer’s disease and cancer [1, 2, 4].Ferroptosis is an iron-dependent form of non-apoptotic cell death induced by excess accumulation of peroxidized phopholipids, generated through oxidation of the PUFA moieties at sn-2 position of membrane phospholipids [5–9]. Ferroptosis is morphologically, biochemically and genetically distinct from other forms of cells death [5], which is tightly regulated by glutathione peroxidase 4 (GPX4) via converting lipid hydroperoxides (PUFA-OOH) into non-toxic lipid alcohols (PUFA-OH) [10, 11]. Emerging evidence indicates that ferroptosis is implicated in ischemia–reperfusion injury (IRI), neurodegeneration, antiviral immunity, cancer immunotherapy and tumor suppression [11–19].Accumulating evidence reveals a robust link between lipid metabolism and ferroptosis [14, 20–24]. However, little is known about the role of ether phospholipids in ferroptosis. In the present study, we revealed the FAR1-TMEM189 axis as a central pathway to drive the susceptibility of ferroptosis. FAR1-TMEM189 axis specifically synthesizes alkyl and vinyl ether phospholipid, where the two isoforms of ether phospholipid play distinct role in ferroptosis. Our findings provide an insight into the mechanism of ether phospholipid-mediated ferroptosis, with implications for novel treatment options for cancer therapy.     

Human-associated microbiota suppress invading bacteria even under disruption by antibiotics

In light of their adverse impacts on resident microbial communities, it is widely predicted that broad-spectrum antibiotics can promote the spread of resistance by releasing resistant strains from competition with other strains and species. We investigated the competitive suppression of a resistant strain of Escherichia coli inoculated into human-associated communities in the presence and absence of the broad and narrow spectrum antibiotics rifampicin and polymyxin B, respectively. We found strong evidence of community-level suppression of the resistant strain in the absence of antibiotics and, despite large changes in community composition and abundance following rifampicin exposure, suppression of the invading resistant strain was maintained in both antibiotic treatments. Instead, the strength of competitive suppression was more strongly associated with the source community (stool sample from individual human donor). This suggests microbiome composition strongly influences the competitive suppression of antibiotic-resistant strains, but at least some antibiotic-associated disruption can be tolerated before competitive release is observed. A deeper understanding of this association will aid the development of ecologically-aware strategies for managing antibiotic resistance.Subject terms: Microbial ecology, Community ecology, Antibiotics

The overuse of broad-spectrum antibiotics in clinical and agricultural settings is a key driver of the current antibiotic resistance crisis [1]. Research into antibiotic resistance has traditionally focused on the evolution of resistance in individual pathogens [2]. In the last decade, researchers have turned their attention to the collateral damage inflicted on commensal members of the microbiome, such as those belonging to the dense communities of the human gastrointestinal tract [3, 4]. Several studies have shown that antibiotics can leave gut communities vulnerable to colonisation by other pathogens [5–7], and, most recently, resistance evolution in invading strains can be facilitated by the absence of community suppression [8, 9]. Taken together, these two lines of enquiry appear to bear out conventional wisdom that relative to narrow-spectrum antibiotics or antibiotic-free conditions, broad spectrum antibiotics should increase the likelihood of communities being invaded by resistant strains [10, 11]. On the other hand, given evidence that community-level properties can sometimes be robust to changes in taxonomic composition [12], it is possible that some antibiotic-associated disruption can be tolerated before colonization resistance is affected. Despite the importance of these contrasting predictions, there have been few, if any, direct tests in human-associated microbiota.We investigated the effect of broad and narrow spectrum antibiotics on the strength of competitive suppression on a resistant variant (generated by in vitro selection for resistance mutations) of a focal strain (Escherichia coli K-12 MG1655) inoculated into gut microbiome communities collected from human faecal samples. The focal strain was jointly resistant to the broad-spectrum antibiotic rifampicin (targets Gram-positives and Gram-negatives via inhibition of the highly conserved bacterial RNA polymerase) and the narrow spectrum antibiotic polymyxin B (only targets Gram-negatives). The focal strain was inoculated alongside live or sterile slurry produced using a sample from one of three healthy human donors (described in [9]) into customized gut media without antibiotics or supplemented with 128 μg/ml rifampicin or 4 μg/ml polymyxin B (see Fig S1). Following 24 h incubation under anaerobic conditions, focal strain density and total biomass were measured via colony counting and flow cytometry, and community composition and diversity were analysed via 16S rRNA sequencing.In the absence of either antibiotic, focal strain density after 24 h was significantly lower in the presence of the three donor communities, indicative of strong competitive suppression (Fig. 1a). Surprisingly, we detected similarly strong competitive suppression in both the antibiotic treatments as we did in the antibiotic-free treatment. Instead, we found that focal strain performance was a stronger function of the specific donor community, irrespective of antibiotic treatment (Figs. 1b, and S2).Open in a separate windowFig. 1Effect of community, donor and antibiotic on focal strain abundance.a Violin plots showing the distribution of observed abundances of the focal strain in each antibiotic treatment. Blue denotes community free treatments; yellow denotes community treatment. Point shape denotes the individual human donor of live community or sterilized slurry: donor 1 = circles, donor 2 = squares, donor 3 = diamonds. b Treatment contrasts (posterior distributions of parameter estimates for a linear model with negative binomial errors) for focal strain abundance as a function of community (live vs sterile slurry), antibiotic (none, polymixin B or rifampicin), and donor (slurry prepared with samples from human donor 1, 2 or 3), and the interactions between community and antibiotic, and community and donor. Posterior parameter estimates in green have 95% credible intervals that do not overlap with 0 (i.e., there is less than 5% probability there is no effect of the variables/interactions captured by these coefficients). The reference level (vertical black line) = donor 1 in the no antibiotic treatment in the absence of the community (i.e., sterilized slurry).What makes these results particularly striking is that, consistent with previous studies [7, 10, 13], treatment with a broad-spectrum antibiotic was still associated with a marked shift in community composition (analysis of 16S amplicon data) (Fig. 2a). Based on OTU composition, all three donors in the rifampicin treatment cluster separately from the polymyxin B and antibiotic-free treatments, which cluster together (Fig. 2b). This divergence in composition appears to be largely driven by enrichment of both Enterobacteriaceae and Erysipelotrichaceae in the rifampicin treatment (Fig. 2a). In addition to strong shifts in composition, total bacterial abundance was significantly reduced in the rifampicin treatment (Figs. 2c and S3). Despite this, total richness and diversity (Shannon Index) after 24 h did not differ between the treatments (Fig. 2c). In contrast, diversity loss over time was more strongly associated with donor identity, with the donor community associated with the weakest competitive suppression (donor 3) also exhibiting the largest decline in richness and diversity across all treatments. This observation is consistent with previous work demonstrating that colonization resistance in the mouse gut is highly contingent on the complexity and composition of the resident microbiota [14].Open in a separate windowFig. 2Community response to antibiotic treatments.a Heatmap of relative abundance of the ten most abundant families of bacteria across treatments (derived from amplicon data). I = inoculum; AB free = Antibiotic free; Poly = polymyxin B; Rif = rifampicin. b NMDS ordination of family level composition in each treatment-donor combination. c Violin plots showing the abundance (top), species richness (middle) and diversity (Shannon Index) (bottom) distributions in each treatment. In b and c: circles = donor 1; squares = donor 2, diamonds = donor 3.A limitation of this study is that we only considered the effects of two antibiotics. Nevertheless, given the scale of community perturbation observed (Fig. 2), we can at least be sure our findings are not explained by a lack of antibiotic effects in our system. There must be some limit dictated by antibiotic concentration, combination, or duration of exposure, beyond which we would expect to observe stronger competitive release. Indeed, prior research has shown that antibiotics can greatly inhibit colonisation resistance [15, 16]. As such, characterizing where this limit lies (e.g., by investigating community-mediated suppression as a function of antibiotic concentration/duration) will be an important challenge for future work. Similarly, although we only considered a single focal strain, and other strains/species may have been more invasive (for example, those with fewer, different or less costly resistance mutations), key for our experiment was that the focal strain had a positive growth rate over the timescale of the experiment, despite exhibiting significant resistance costs in antibiotic-free assays (Fig. S1). This allowed us to test for sensitivity of competitive suppression to antibiotic treatment. We also note that in spite of a small boost in the focal strain’s performance in the presence of rifampicin independent of the community (a possible hormetic response [17] absent under aerobic growth in LB, Fig S1), we did not observe an increase in the magnitude of competitive release in the rifampicin treatment. Finally, the drop in diversity indicates, unsurprisingly, microcosms are a novel environment relative to the source environment. Despite this, key taxa in each community were stable over the course of the experiment, and previously over a longer timescale in the same set-up [9], demonstrating these conditions sustain diverse human-associated communities over relevant timescales.In conclusion, these results are consistent with prevailing wisdom that healthy gut communities can suppress invading strains and thereby reduce the likelihood of resistance emerging [8, 9, 18]. Nevertheless, the absence of a significant effect of broad, or even narrow, spectrum antibiotics on the degree of competitive suppression of our focal strain is much more surprising. Despite the limitations of scope discussed above, this shows that the functional diversity of gut communities may be more robust to disturbance by broad spectrum antibiotics than previously recognised. This is not to suggest that the use of broad-spectrum antibiotics does not drive marked changes in composition but rather that there is some degree of functional redundancy in diverse communities that facilitates the maintenance of competitive suppression [12, 19]. Notwithstanding the need to test how these findings translate to in vivo settings, this finding is relevant for optimizing personalised treatments that either account for disruption by antibiotics or that make microbiomes harder for pathogens to invade.     

A continuum mechanics model for the Fåhræus-Lindqvist effect

The decrease in apparent relative viscosity that occurs when blood is made to flow through a tube whose diameter is less than about 0.3 mm is a well-known and documented phenomenon in physiology, known as the Fåhræus-Lindqvist effect. However, since the historical work of Fåhræus and Lindqvist (Amer. J. Physiol. 96(3): pp. 562–568, 1931), the underlying physical mechanism has remained enigmatic. A widely accepted qualitative explanation was provided by Haynes (Amer. J. Physiol. 198, pp. 1193–1200, 1960) according to which blood flows in microvessels with a core-annulus structure, where the erythrocytes concentrate within a central core surrounded by a plasma layer. Although sustained by observations, this conjecture lacks a rigorous deduction from the basic principles of continuum dynamics. Moreover, relations aimed to reproduce the blood apparent relative viscosity, extensively used in micro-circulation, are all empirical and not derived from the analysis of the fluid mechanical phenomena involved. In this paper, we apply the recent results illustrated in Guadagni and Farina (Int. J. Nonlinear Mech. 126, p. 103587, 2020), with the purpose of showing that Haynes’ conjecture, slightly corrected to make it more realistic, can be proved and can be used to reach a sound explanation of the Fåhræus-Lindqvist effect based on continuum mechanics. We propose a theoretical model for the blood apparent relative viscosity which is validated by matching not only the original experimental data reported by Fåhræus and Lindqvist (Amer. J. Physiol. 96(3), pp. 562–568, 1931), but also those provided by several subsequent authors.     

Reduction of the 2,2′-Azinobis(3-Ethylbenzthiazoline-6-Sulfonate) Cation Radical by Physiological Organic Acids in the Absence and Presence of Manganese

Laccase is a copper-containing phenoloxidase, involved in lignin degradation by white rot fungi. The laccase substrate range can be extended to include nonphenolic lignin subunits in the presence of a noncatalytic cooxidant such as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), with ABTS being oxidized to the stable cation radical, ABTS^·+, which accumulates. In this report, we demonstrate that the ABTS^·+ can be efficiently reduced back to ABTS by physiologically occurring organic acids such as oxalate, glyoxylate, and malonate. The reduction of the radical by oxalate results in the formation of H₂O₂, indicating the formation of O₂^·− as an intermediate. O₂^·− itself was shown to act as an ABTS^·+ reductant. ABTS^·+ reduction and H₂O₂ formation are strongly stimulated by the presence of Mn²⁺, with accumulation of Mn³⁺ being observed. Additionally, 4-methyl-O-isoeugenol, an unsaturated lignin monomer model, is capable of directly reducing ABTS^·+. These data suggest several mechanisms for the reduction of ABTS^·+ which would permit the effective use of ABTS as a laccase cooxidant at catalytic concentrations.Lignin, the second most abundant renewable organic compound in the biosphere after cellulose, is highly recalcitrant, and therefore its biodegradation is a rate-limiting step in the global carbon cycle (9). White rot fungi have evolved a unique mechanism to accomplish this degradation, which utilizes extracellular enzymes to generate oxidative radical species (16). This degradative system is highly nonspecific, and as a consequence, these fungi can also oxidize a broad spectrum of structurally diverse environmental pollutants (4, 18). Three main groups of enzymes, i.e., lignin peroxidases (LiP), manganese peroxidases (MnP), and laccases, along with their low-molecular-weight cofactors, have been implicated in the lignin degradation process. LiP can oxidize the nonphenolic aromatic moieties that make up approximately 85% of the lignin polymer (21), while MnP uses the Mn²⁺/Mn³⁺ couple to oxidize phenolic subunits (19). Laccase, a copper-containing phenoloxidase, catalyzes the four-electron reduction of oxygen to water, and this is accompanied by the oxidation of a phenolic substrate (32).In recent years, however, the laccase substrate range has been extended to include nonphenolic lignin subunits in the presence of readily oxidizable primary substrates. These cooxidants have been denoted mediators because they were previously speculated (but not proven) to act as electron transfer mediators. The most extensively investigated laccase mediator is 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), a synthetic nitrogen-substituted aromatic compound which allows the oxidation of nonphenolic lignin model compounds (6) and the delignification of kraft pulp (8) by laccase. More recent work has also focused on an alternative compound, 1-hydroxybenzotriazole (7, 10). In the presence of these compounds, laccase can also catalyze the oxidation of polycyclic aromatic hydrocarbons (PAH) (12, 23), chemical synthesis (29), and textile dye bleaching (31). ABTS is oxidized by laccase to its corresponding cation radical. In the case of ABTS, the radical (ABTS^·+) is highly stable, and it has been suggested that it may act as a diffusible oxidant of the enzyme (7). However, although the redox chemistry of ABTS (22) and its radical has been characterized, the mechanisms by which it interacts with laccase to “mediate” lignin oxidation are still unknown. Potthast et al. (28) have found evidence suggesting that ABTS acts as an activator or cooxidant of the enzyme. The observation that the laccase/ABTS couple can oxidize the nonphenolic veratryl alcohol, while ABTS^·+ alone cannot (6), provides a further indication of this activator role for ABTS. If compounds such as ABTS do indeed act as cooxidants of the enzyme, it is necessary that some mechanism(s) exists for the recycling of their cation radicals back to their reduced forms so as to be available for subsequent catalytic cycles.A number of low-molecular-weight compounds have been implicated in the catalysis of MnP during the oxidation of lignin. The most important of these is manganese, which is present in virtually all woody tissues (17). Divalent manganese (Mn²⁺) is oxidized by the enzyme to the trivalent form (Mn³⁺), which is capable of oxidizing an extensive range of phenolic compounds (19). To catalyze lignin oxidation, Mn³⁺ is chelated and stabilized by organic acids, which facilitate its diffusion to act as an oxidant at a distance from the MnP active site (19, 33). A range of these acids are produced by ligninolytic fungi (25, 30, 33), but the most ubiquitous is oxalate, whose production at levels as high as 28 mM by cultures of Pleurotus ostreatus has been observed (1). Oxalate can itself be oxidized by Mn³⁺, producing the formate anion radical (CO₂^·−), which can then reduce molecular oxygen to produce superoxide (O₂^·−) (24), and a role for these radicals as reducing agents in lignin degradation has been suggested (24).In this report, evidence is presented indicating that physiologically occurring organic acids can directly reduce ABTS^·+. The rate of reduction is highly stimulated by the presence of manganese, and the results indicate a mechanism involving O₂^·−.     

Phospholamban phosphorylation,mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy

We review the recent development of novel biochemical and spectroscopic methods to determine the site-specific phosphorylation, expression, mutation, and structural dynamics of phospholamban (PLB), in relation to its function (inhibition of the cardiac calcium pump, SERCA2a), with specific focus on cardiac physiology, pathology, and therapy. In the cardiomyocyte, SERCA2a actively transports Ca²⁺ into the sarcoplasmic reticulum (SR) during relaxation (diastole) to create the concentration gradient that drives the passive efflux of Ca²⁺ required for cardiac contraction (systole). Unphosphorylated PLB (U-PLB) inhibits SERCA2a, but phosphorylation at S16 and/or T17 (producing P-PLB) changes the structure of PLB to relieve SERCA2a inhibition. Because insufficient SERCA2a activity is a hallmark of heart failure, SERCA2a activation, by gene therapy (Andino et al. 2008; Fish et al. 2013; Hoshijima et al. 2002; Jessup et al. 2011) or drug therapy (Ferrandi et al. 2013; Huang 2013; Khan et al. 2009; Rocchetti et al. 2008; Zhang et al. 2012), is a widely sought goal for treatment of heart failure. This review describes rational approaches to this goal. Novel biophysical assays, using site-directed labeling and high-resolution spectroscopy, have been developed to resolve the structural states of SERCA2a-PLB complexes in vitro and in living cells. Novel biochemical assays, using synthetic standards and multidimensional immunofluorescence, have been developed to quantitate PLB expression and phosphorylation states in cells and human tissues. The biochemical and biophysical properties of U-PLB, P-PLB, and mutant PLB will ultimately resolve the mechanisms of loss of inhibition and gain of inhibition to guide therapeutic development. These assays will be powerful tools for investigating human tissue samples from the Sydney Heart Bank, for the purpose of analyzing and diagnosing specific disorders.     

Size-Selective Predation on Groundwater Bacteria by Nanoflagellates in an Organic-Contaminated Aquifer

Time series incubations were conducted to provide estimates for the size selectivities and rates of protistan grazing that may be occurring in a sandy, contaminated aquifer. The experiments involved four size classes of fluorescently labeled groundwater bacteria (FLB) and 2- to 3-μm-long nanoflagellates, primarily Spumella guttula (Ehrenberg) Kent, that were isolated from contaminated aquifer sediments (Cape Cod, Mass.). The greatest uptake and clearance rates (0.77 bacteria · flagellate⁻¹ · h⁻¹ and 1.4 nl · flagellate⁻¹ · h⁻¹, respectively) were observed for 0.8- to 1.5-μm-long FLB (0.21-μm³ average cell volume), which represent the fastest growing bacteria within the pore fluids of the contaminated aquifer sediments. The 19:1 to 67:1 volume ratios of nanoflagellate predators to preferred bacterial prey were in the lower end of the range commonly reported for other aquatic habitats. The grazing data suggest that the aquifer nanoflagellates can consume as much as 12 to 74% of the unattached bacterial community in 1 day and are likely to have a substantive effect upon bacterial degradation of organic groundwater contaminants.While heterotrophic protists have been found in pristine and contaminated aquifers (3, 29, 34, 37, 54–57), very little research has been performed to elucidate their role in the subsurface. In other environments (e.g., surface and marine waters, topsoil, and wastewater treatment plants), it is well documented that they typically consume bacteria (2, 11, 15, 41, 42, 47), although some have been observed to consume high-molecular-weight organics (48, 59) and even viruses (20, 39). Protists typically graze selectively, depending upon the size (1, 9, 17, 25, 52), growth condition (18, 53), species (16, 17, 35), and motility (18) of their prey. In carbon-limited environments, protists decrease bacterial competition, resulting in a greater bacterial uptake rate for organic substrate per unit of bacterial biomass (27). Based upon indirect field observations, it is also hypothesized that this may be the role they play in organically contaminated aquifers (31). In nutrient-limited environments, protists may release nitrogen or phosphorus needed by bacteria (10, 28, 44, 61).Studies at the U.S. Geological Survey’s (USGS) Toxic Substances Hydrology Program research site at the Massachusetts Military Reservation (MMR) on Cape Cod, Mass., have shown that sandy aquifer sediments can harbor large protistan populations even at relatively low levels (≤2 mg/liter) of dissolved organic matter (30). Protistan abundances in the MMR aquifer plume range from 1 × 10⁴ to 7 × 10⁴ g (dry weight)⁻¹ (30) and consist primarily of nanoflagellates (2 to 3 μm in length) (29) that belong to the genera Bodo, Cercomonas, Cryptaulax, Cyanthomonas, Goniomonas, and Spumella, along with some undescribed species (37). A few amoebae (63) and no ciliates (29) have been observed.Results of a principal-component factor analysis of protistan and bacterial abundances and chemical constituents in the MMR plume suggested that the flagellates were preying upon unattached bacteria (30). Additional evidence of predation was obtained from flowthrough columns of aquifer sediment from which fluorescently labeled unattached bacteria eluted at much lower rates than they did from sterile (protist-free) controls (31). However, these results provide only indirect evidence of predation because no enumerations of the bacteria within the flagellates were performed.The purpose of the research reported in this paper was to directly determine whether the MMR nanoflagellates can consume unattached bacteria in the plume and the extent to which they engage in size-selective grazing. Rates of bacterivory (grazing and clearance rates) were estimated in the laboratory by using fluorescently labeled, monodispersed bacteria (FLB) and nanoflagellates that had been isolated from the MMR aquifer plume. Although other methods exist (19, 26, 38, 43, 45, 62, 64–66), we chose to use fluorescent labeling to study flagellate bacterivory because this procedure requires shorter incubation times and relies upon direct visual observation of the prey within the predator. In addition, experiments could be designed with different sizes of FLB to determine if the nanoflagellates can discriminate between prey. This involved using FLB with cell lengths of 0.1 to 0.5, 0.5 to 0.8, 0.8 to 1.5, and >1.5 μm (average cell volumes of 0.06, 0.14, 0.21, and 0.87 μm³, respectively) in the grazing experiments.     

CO2 Uptake and Fixation by a Thermoacidophilic Microbial Community Attached to Precipitated Sulfur in a Geothermal Spring

Carbon fixation at temperatures above 73°C, the upper limit for photosynthesis, is carried out by chemosynthetic thermophiles. Yellowstone National Park (YNP), Wyoming possesses many thermal features that, while too hot for photosynthesis, presumably support chemosynthetic-based carbon fixation. To our knowledge, in situ rates of chemosynthetic reactions at these high temperatures in YNP or other high-temperature terrestrial geothermal springs have not yet been reported. A microbial community attached to precipitated elemental sulfur (S^o floc) at the source of Dragon Spring (73°C, pH 3.1) in Norris Geyser Basin, YNP, exhibited a maximum rate of CO₂ uptake of 21.3 ± 11.9 μg of C 10⁷ cells⁻¹ h⁻¹. When extrapolated over the estimated total quantity of S^o floc at the spring''s source, the S^o floc-associated microbial community accounted for the uptake of 121 mg of C h⁻¹ at this site. On a per-cell basis, the rate was higher than that calculated for a photosynthetic mat microbial community dominated by Synechococcus spp. in alkaline springs at comparable temperatures. A portion of the carbon taken up as CO₂ by the S^o floc-associated biomass was recovered in the cellular nucleic acid pool, demonstrating that uptake was coupled to fixation. The most abundant sequences in a 16S rRNA clone library of the S^o floc-associated community were related to chemolithoautotrophic Hydrogenobaculum strains previously isolated from springs in the Norris Geyser Basin. These microorganisms likely contributed to the uptake and fixation of CO₂ in this geothermal habitat.The upper temperature limit for primary production via photosynthesis is ∼73°C (7, 8, 11). At this temperature, photosynthesis is restricted to cyanobacteria of the genus Synechococcus, which generally inhabit alkaline environments (11). In acidic environments (pH < 4.0), the upper temperature limit for photosynthetic-based primary production is ∼56°C. Under these conditions, phototrophic activity is restricted to the unicellular eukaryotic red algae Cyanidium, Galdieria, and Cyanidioschyzon, collectively referred to as “cyanidia” (6, 12, 31, 48). Primary production above this temperature in acidic environments occurs through chemoautotrophy, a metabolism restricted to prokaryotes.Yellowstone National Park (YNP), WY, possesses numerous high-temperature (73 to 93°C) geothermal environments that are thought to support communities of microorganisms through chemoautotrophic-based primary production. Evidence for chemosynthesis in these environments is based on the recovery of 16S rRNA gene sequences that are affiliated with cultivated representatives of the phyla Aquificae and Crenarchaeota, many of which are capable of CO₂ fixation via the oxidation of hydrogen (H₂) and/or sulfide (HS⁻) (15, 17, 21, 24, 26, 28, 41, 46). Surprisingly, CO₂ fixation has yet to be demonstrated in situ in YNP hot spring environments (acidic or alkaline) where temperatures exceed the limits of photosynthesis and where primary production is thought to be driven by chemoautotrophic metabolism (14, 15, 28, 29).Dragon Spring, an acid-sulfate-chloride (ASC) spring located in the Norris Geyser Basin of YNP, is a likely habitat for chemoautotrophic primary production. The pH of the water is ∼3.1, and the temperature of the water at the source fluctuates from 65 to 78°C, which is well above the upper temperature limit for photosynthesis under acidic conditions. Potential electron donors for chemolithoautotrophic growth in the source water include hydrogen (H₂) and sulfide (S²⁻) at concentrations of 13 nM and 65 μM, respectively (15). In addition, submerged substrata at the spring''s source are blanketed by precipitates of elemental sulfur (S°), hereafter referred to as S^o floc (23). Inventories of bacterial and archaeal 16S rRNA genes recovered from S^o floc collected from the source of Dragon Spring indicate the presence of Crenarchaeota and Aquificae (4, 15). The latter are related to chemolithoautotrophic Hydrogenobaculum spp., representatives of which have recently been isolated from the spring (15). In the present study, we demonstrate uptake and fixation of CO₂ at a temperature of 73°C by a Hydrogenobaculum-dominated microbial community associated with S^o floc collected from the source of Dragon Spring. This is the first direct evidence of CO₂ uptake in situ by a thermoacidophilic microbial community at a temperature that precludes photosynthesis in terrestrial geothermal springs.     

Temperature Responses of C4 Photosynthesis: Biochemical Analysis of Rubisco,Phosphoenolpyruvate Carboxylase,and Carbonic Anhydrase in Setaria viridis

The photosynthetic assimilation of CO₂ in C₄ plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C₄ biochemical models of leaf CO₂ exchange in response to changes in CO₂ availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C₄ plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO₃⁻, and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C₄ plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO₃⁻ limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C₃ species and the C₄ plant S. viridis.Biochemical models of photosynthesis are often used to predict the effect of environmental conditions on net rates of leaf CO₂ assimilation (Farquhar et al., 1980; von Caemmerer, 2000, 2013; Walker et al., 2013). With climate change, there is increased interest in modeling and understanding the effects of changes in temperature and CO₂ concentration on photosynthesis. The biochemical models of photosynthesis are primarily driven by the kinetic properties of the enzyme Rubisco, the primary carboxylating enzyme of the C₃ photosynthetic pathway, catalyzing the reaction of ribulose-1,5-bisphosphate (RuBP) with either CO₂ or oxygen. However, the CO₂-concentrating mechanism in C₄ photosynthesis utilizes carbonic anhydrase (CA) to help maintain the chemical equilibrium of CO₂ with HCO₃⁻ and phosphoenolpyruvate carboxylase (PEPc) to catalyze the carboxylation of phosphoenolpyruvate (PEP) with HCO₃⁻. These reactions ultimately provide the elevated levels of CO₂ to the compartmentalized Rubisco (Edwards and Walker, 1983). In C₄ plants, it has been demonstrated that PEPc, Rubisco, and CA can limit rates of CO₂ assimilation and influence the efficiency of the CO₂-concentrating mechanism (von Caemmerer, 2000; von Caemmerer et al., 2004; Studer et al., 2014). Therefore, accurate modeling of leaf photosynthesis in C₄ plants in response to future climatic conditions will require temperature parameterizations of Rubisco, PEPc, and CA kinetics from C₄ species.Modeling C₄ photosynthesis relies on the parameterization of both PEPc and Rubisco kinetics, making it more complex than for C₃ photosynthesis (Berry and Farquhar, 1978; von Caemmerer, 2000). However, the activity of CA is not included in these models, as it is assumed to be nonlimiting under most conditions (Berry and Farquhar, 1978; von Caemmerer, 2000). This assumption is implemented by modeling PEPc kinetics as a function of CO₂ partial pressure (pCO₂) and not HCO₃⁻ concentration, assuming CO₂ and HCO₃⁻ are in chemical equilibrium. However, there are questions regarding the amount of CA activity needed to sustain rates of C₄ photosynthesis and if CO₂ and HCO₃⁻ are in equilibrium (von Caemmerer et al., 2004; Studer et al., 2014).The most common steady-state biochemical models of photosynthesis are derived from the Michaelis-Menten models of enzyme activity (von Caemmerer, 2000), which are driven by the V_max and the K_m. Both of these parameters need to be further described by their temperature responses to be used to model photosynthesis in response to temperature. However, the temperature response of plant CA activity has not been completed above 17°C, and there is no known measured temperature response of K_m HCO₃⁻ for PEPc (K_P). Alternatively, Rubisco has been well studied, and there are consistent differences in kinetic values between C₃ and C₄ species at 25°C (von Caemmerer and Quick, 2000; Kubien et al., 2008), but the temperature responses, including both carboxylation and oxygenation reactions, have only been performed in C₃ species (Badger and Collatz, 1977; Jordan and Ogren, 1984; Bernacchi et al., 2001, 2002; Walker et al., 2013).Here, we present the temperature dependency of Rubisco carboxylation and oxygenation reactions, PEPc kinetics for HCO₃⁻, and CA hydration from 10°C to 40°C from the C₄ species Setaria viridis (succession no., A-010) measured using membrane inlet mass spectrometry. Generally, the 25°C values of the Rubisco parameters were similar to previous measurements of C₄ species. The temperature response of the maximum rate of Rubisco carboxylation (V_cmax) was high compared with most previous measurements from both C₃ and C₄ species, and the temperature response of the K_m for oxygenation (K_O) was low compared with most previously measured species. Taken together, the modeled temperature responses of Rubisco activity in S. viridis were similar to the previously reported temperature responses of some C₃ species. Additionally, the temperature response of the maximum rate of PEPc carboxylation (V_pmax) was similar to previous measurements. However, the temperature response of K_P was lower than what has been predicted (Chen et al., 1994). For CA, deactivation of the hydration activity was observed above 25°C. Additionally, models of CA and PEPc show that CA activity limits HCO₃⁻ availability to PEPc above 15°C, suggesting that CA limits PEP carboxylation rates in S. viridis when compared with the assumption that CO₂ and HCO₃⁻ are in full chemical equilibrium.