Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

Hyaluronic acid (HA) is a high‐value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust “Generally Recognized as Safe (GRAS)” organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase (HAS) of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production. Elevation of membrane CL levels via overexpressing components involved in the CL biosynthesis pathway, and redistribution of CL along the lateral membrane via repression of the cell division initiator protein FtsZ resulted in increases to the HA titer of up to 204% and peak molecular weight of up to 2.2 MDa. Moreover, removal of phosphatidylethanolamine and neutral glycolipids from the membrane of HA‐producing B. subtilis via inactivation of pssA and ugtP, respectively, has suggested the lipid dependence for functional expression of SeHAS. Our study demonstrates successful application of membrane engineering strategies to develop an effective platform for biomanufacturing of HA with B. subtilis strains expressing Class I streptococcal HAS.     

Risk and protective factors for mental disorders beyond genetics: an evidence‐based atlas

Decades of research have revealed numerous risk factors for mental disorders beyond genetics, but their consistency and magnitude remain uncer­tain. We conducted a “meta‐umbrella” systematic synthesis of umbrella reviews, which are systematic reviews of meta‐analyses of individual studies, by searching international databases from inception to January 1, 2021. We included umbrella reviews on non‐purely genetic risk or protective factors for any ICD/DSM mental disorders, applying an established classification of the credibility of the evidence: class I (convincing), class II (highly suggestive), class III (suggestive), class IV (weak). Sensitivity analyses were conducted on prospective studies to test for temporality (reverse causation), TRANSD criteria were applied to test transdiagnosticity of factors, and A Measurement Tool to Assess Systematic Reviews (AMSTAR) was employed to address the quality of meta‐analyses. Fourteen eligible umbrella reviews were retrieved, summarizing 390 meta‐analyses and 1,180 associations between putative risk or protective factors and mental disorders. We included 176 class I to III evidence associations, relating to 142 risk/protective factors. The most robust risk factors (class I or II, from prospective designs) were 21. For dementia, they included type 2 diabetes mellitus (risk ratio, RR from 1.54 to 2.28), depression (RR from 1.65 to 1.99) and low frequency of social contacts (RR=1.57). For opioid use disorders, the most robust risk factor was tobacco smoking (odds ratio, OR=3.07). For non‐organic psychotic disorders, the most robust risk factors were clinical high risk state for psychosis (OR=9.32), cannabis use (OR=3.90), and childhood adversities (OR=2.80). For depressive disorders, they were widowhood (RR=5.59), sexual dysfunction (OR=2.71), three (OR=1.99) or four‐five (OR=2.06) metabolic factors, childhood physical (OR=1.98) and sexual (OR=2.42) abuse, job strain (OR=1.77), obesity (OR=1.35), and sleep disturbances (RR=1.92). For autism spectrum disorder, the most robust risk factor was maternal overweight pre/during pregnancy (RR=1.28). For attention‐deficit/hyperactivity disorder (ADHD), they were maternal pre‐pregnancy obesity (OR=1.63), maternal smoking during pregnancy (OR=1.60), and maternal overweight pre/during pregnancy (OR=1.28). Only one robust protective factor was detected: high physical activity (hazard ratio, HR=0.62) for Alzheimer’s disease. In all, 32.9% of the associations were of high quality, 48.9% of medium quality, and 18.2% of low quality. Transdiagnostic class I‐III risk/protective factors were mostly involved in the early neurodevelopmental period. The evidence‐based atlas of key risk and protective factors identified in this study represents a benchmark for advancing clinical characterization and research, and for expanding early intervention and preventive strategies for mental disorders.     

Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors

Different isoforms of serotonin subtype 2C receptor (5-HT(2C)R) with altered G protein-coupling efficacy are generated by RNA editing, which converts genomically encoded adenosine residues into inosines. In combination, editing of five sites all located within the second intracellular loop region of 5-HT(2C)R mRNA changes the gene-encoded Ile, Asn, and Ile at positions 156, 158, and 160, respectively. We analyzed the G protein-coupling functions of previously unreported editing isoform receptors. An approximately 13-fold reduction in the agonist potency for G protein-coupling stimulation as well as a significantly reduced basal level activity was observed with the thalamus-specific isoform carrying Ile156, Gly158, and Val160 (5-HT(2C)R-IGV). In contrast, the agonist was four- to five-fold less potent with 5-HT(2C)R-MSV and -IDV, detected in the amygdala and choroid plexus, respectively, indicating a dominant role for the amino acid residue at position 158 in receptor functions. We also identified a splicing variant receptor with a truncated C terminus that displayed no ligand binding capacity or G protein-coupling activity. Examination of the alternatively spliced RNA encoding this truncated receptor suggests that editing of this variant RNA occurs after completion of splicing, resulting in complete editing at all five sites.     

禽流感H5N1病毒感染BALB/c小鼠后多器官病变初探

为了研究禽流感H5N1病毒在各个器官的增殖和病理变化,在生物安全实验室,我们将禽流感H5N1病毒通过尾静脉接种BALB/C小鼠。结果小鼠在不经过适应的情况下,直接感染发病,甚至死亡。在观察的7天内,感染小鼠临床症状主要表现呼吸急促,体温、体重下降。尸检表现肺出血,心外膜坏死以及肝脏的坏死。组织病理检查表现心、肝、肺等多器官的病变。肺的病变伴有纤维化的弥漫性肺泡损伤;心肌外膜大量淋巴细胞浸润、坏死;肝细胞大量坏死,淋巴细胞浸润。心、肝的坏死病变在H5N1禽流感病毒相关的研究中未见报道。经过对各个组织器官的病毒载量的检测,未发现病毒在各个病变组织中的复制。免疫组化的检测,各个组织中也未检出阳性的细胞反应。因此,我们认为H5N1禽流感病毒感染小鼠引起多个器官组织的损伤,甚至死亡,不是病毒在器官的复制,而可能是病毒感染小鼠,产生炎症细胞因子的高度表达,损伤多个器官组织所致。     

Toward an optimal sampling protocol for Hemiptera on understorey plants

There are no standardised sampling protocols for inventorying Hemiptera from understorey or canopy plants. This paper proposes an optimal protocol for the understorey, after evaluating the efficiency of seven methods to maximise the richness of Hemiptera collected from plants with minimal field and laboratory time. The methods evaluated were beating, chemical knockdown, sweeping, branch clipping, hand collecting, vacuum sampling and sticky trapping. These techniques were tested at two spatial scales: 1 ha sites and individual plants. In addition, because efficiency may differ with vegetation structure, sampling of sites was conducted in three disparate understorey habitats, and sampling of individual plants was conducted across 33 plant species. No single method sampled the majority of hemipteran species in the understorey. Chemical knockdown, vacuum sampling and beating yielded speciose samples (61, 61 and 30 species, respectively, representing 53, 53 and 26% of total species collected). The four remaining methods provided species-poor samples (<18 species or <16% of total species collected). These methods also had biases towards particular taxa (e.g., branch clipping and hand collecting targeted sessile Hemiptera, and sticky trapping were dominated by five species of Psyllidae). The most time-efficient methods were beating, sweeping and hand collecting (200 minutes of field and laboratory time yielded >7 species for each technique). By comparison, vacuum sampling, sticky trapping, branch clipping and chemical knockdown yielded <5 species for the same period. Chemical knockdown had further disadvantages; high financial cost and potential spray drift. The most effective methods for a standardised sampling protocol to inventory Hemiptera from the understorey are beating and vacuum sampling. If used in combination, these methods optimise the catch of understorey hemipteran species, as their samples have high complementarity.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 degrees C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

Persistent maternal effects on juvenile survival in North American red squirrels

Maternal effects can have lasting fitness consequences for offspring, but these effects are often difficult to disentangle from associated responses in offspring traits. We studied persistent maternal effects on offspring survival in North American red squirrels (Tamiasciurus hudsonicus) by manipulating maternal nutrition without altering the post-emergent nutritional environment experienced by offspring. This was accomplished by providing supplemental food to reproductive females over winter and during reproduction, but removing the supplemental food from the system prior to juvenile emergence. We then monitored juvenile dispersal, settlement and survival from birth to 1 year of age. Juveniles from supplemented mothers experienced persistent and magnifying survival advantages over juveniles from control mothers long after supplemental food was removed. These maternal effects on survival persisted, despite no observable effect on traits normally associated with high offspring quality, such as body size, dispersal distance or territory quality. However, supplemented mothers did provide their juveniles an early start by breeding an average of 18 days earlier than control mothers, which may explain the persistent survival advantages their juveniles experienced.     

Mapping a new spontaneous preterm birth susceptibility gene, IGF1R, using linkage, haplotype sharing, and association analysis

Preterm birth is the major cause of neonatal death and serious morbidity. Most preterm births are due to spontaneous onset of labor without a known cause or effective prevention. Both maternal and fetal genomes influence the predisposition to spontaneous preterm birth (SPTB), but the susceptibility loci remain to be defined. We utilized a combination of unique population structures, family-based linkage analysis, and subsequent case-control association to identify a susceptibility haplotype for SPTB. Clinically well-characterized SPTB families from northern Finland, a subisolate founded by a relatively small founder population that has subsequently experienced a number of bottlenecks, were selected for the initial discovery sample. Genome-wide linkage analysis using a high-density single-nucleotide polymorphism (SNP) array in seven large northern Finnish non-consanginous families identified a locus on 15q26.3 (HLOD 4.68). This region contains the IGF1R gene, which encodes the type 1 insulin-like growth factor receptor IGF-1R. Haplotype segregation analysis revealed that a 55 kb 12-SNP core segment within the IGF1R gene was shared identical-by-state (IBS) in five families. A follow-up case-control study in an independent sample representing the more general Finnish population showed an association of a 6-SNP IGF1R haplotype with SPTB in the fetuses, providing further evidence for IGF1R as a SPTB predisposition gene (frequency in cases versus controls 0.11 versus 0.05, P = 0.001, odds ratio 2.3). This study demonstrates the identification of a predisposing, low-frequency haplotype in a multifactorial trait using a well-characterized population and a combination of family and case-control designs. Our findings support the identification of the novel susceptibility gene IGF1R for predisposition by the fetal genome to being born preterm.     

Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.     

Spermatogenesis in the turkey (Meleagris gallopavo): quantitative approach in immature and adult males subjected to various photoperiods

The objectives of this study were to identify and quantitate the germ cell populations of the testes in sexually mature male turkeys (Trial 1), determine the duration of meiosis based on BrdU labeling and stereological analyses (Trial 2), and examine the impact of various photoperiods on germinal and somatic cell populations in immature and adult males (Trial 3). In Trial 1, both testes within a male had similar stereological components (P>0.05) for all parameters analyzed. In Trial 2, the duration of Type-1 spermatocytes and round spermatids in turkeys lasted 4.5+/-0.5 and 2.0+/-0.5 days, respectively. In Trial 3, the short photoperiod (7L:17D) delayed testicular growth (in the stereological parameters analyzed). In contrast, the effect of a moderately short photoperiod (10.5L:13.5D) was comparable to the effect of a long (14L:10D) or increasing photoperiod (7L:17D to 14L:10D) on the stereological parameters examined. With the exception of the short photoperiod, all other photoperiods used in this study induced comparable early testicular maturation, with maximum testis weight at 29-35 weeks of age. As the males got older, there was a progressive, linear decline in testis weight through 60 weeks, at which time there were no significant differences among photoperiods. In conclusion, the duration of meiosis in the turkey was similar to that observed in the fowl and guinea-fowl. The existence of a threshold of photosensitivity to gonad stimulation in male turkeys is suggested to be between 7.0 and 10.5 h of light.