Deciphering mechanisms and implications of bacterial translocation in human health and disease

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Natural products in antibiotic development: is the success story over?

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CARD9 gene rs4077515 polymorphism is associated with the susceptibility of Hashimoto’s thyroiditis and the development of thyroid cancer

AmisHashimoto’s thyroiditis (HT) is the most common type of autoimmune thyroiditis and is a risk factor for the occurrence of thyroid papillary carcinoma (PTC). The study aimed to explore the distribution of CARD9 rs4077515 polymorphism in HT and PTC patients, in order to evaluate its association with the occurrence and development of HT.Methods150 HT patients and 120 PTC cases were included. Genotypes of CARD9 rs40775155 polymorphism were sequenced and counted.ResultsA remarkable increase trend of rs4077515 AA genotype was found in HT cases in comparison with the control group, while GG genotype frequency exhibited a down trend. An excess of A allele was also detected in HT group. HT cases carrying AG and AA genotypes had high risk to receive hormonotherapy and needed a much larger dose. In comparison with HT cases, both AG and AA appeared more frequently in PTC patients, and are associated with the tumor size, LN metastasis and surgical margin. The AG (OR = 2.566, 95 % CI = 1.376–4.786) and AA (OR = 3.040, 95 % CI = 1.525–6.060) genotype carriers had a greater risk of developing PTC. The A allele of rs4077515 polymorphism was a risk allele for the onset of PTC among HT cases (OR = 1.775, 95 % CI = 1.260–2.502).ConclusionCARD9 rs4077515 polymorphism is likely to be a risk factor for HT in the Chinese Han population, it also contributes to the development of PTC for HT patients.     

Impact on S. aureus and E. coli Membranes of Treatment with Chlorhexidine and Alcohol Solutions: Insights from Molecular Simulations and Nuclear Magnetic Resonance

Membranes form the first line of defence of bacteria against potentially harmful molecules in the surrounding environment. Understanding the protective properties of these membranes represents an important step towards development of targeted anti-bacterial agents such as sanitizers. Use of propanol, isopropanol and chlorhexidine can significantly decrease the threat imposed by bacteria in the face of growing anti-bacterial resistance via mechanisms that include membrane disruption. Here we have employed molecular dynamics simulations and nuclear magnetic resonance to explore the impact of chlorhexidine and alcohol on the S. aureus cell membrane, as well as the E. coli inner and outer membranes. We identify how sanitizer components partition into these bacterial membranes, and show that chlorhexidine is instrumental in this process.     

Dhh signaling pathway regulates reconstruction of seminiferous tubule-like structure

The reconstruction of a tubule-like structure in vitro has provided a promising system to analyze factors that drive morphogenesis and the underlying mechanisms. In this study, we took advantage of the inhibitor cyclopamine and a smoothened agonist to detect the role of the Dhh signaling pathway in the reconstructed tubule-like structure. Sertoli cells did not show polarity, rounded peritubular myoid cells and scattered Leydig cells were observed, combined with less laminin and lower proliferation of Leydig, peritubular myoid, germ, and Sertoli cells. However, in the presence of SAG, elongated peritubular myoid cells gathered at the bottom of polarized Sertoli cells, and most of the Leydig cells gathered at the outer part of the elongated peritubular myoid cells. Moreover, SAG promoted the secretion of laminin, assisting in the formation of the basal membrane and promoting the proliferation of Leydig, peritubular myoid, and germ cells. The level of Gli1 was significantly downregulated when treated with cyclopamine, whereas it was significantly upregulated when treated with SAG. These results indicate that the Dhh signaling pathway regulates the reconstruction of tubule-like structures by regulating the expression of Gli1.     

Continuous biomanufacturing with microbes — upstream progresses and challenges

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A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.     

A mechanistic protrusive-based model for 3D cell migration

Cell migration is essential for a variety of biological processes, such as embryogenesis, wound healing, and the immune response. After more than a century of research—mainly on flat surfaces—, there are still many unknowns about cell motility. In particular, regarding how cells migrate within 3D matrices, which more accurately replicate in vivo conditions. We present a novel in silico model of 3D mesenchymal cell migration regulated by the chemical and mechanical profile of the surrounding environment. This in silico model considers cell’s adhesive and nuclear phenotypes, the effects of the steric hindrance of the matrix, and cells ability to degradate the ECM. These factors are crucial when investigating the increasing difficulty that migrating cells find to squeeze their nuclei through dense matrices, which may act as physical barriers. Our results agree with previous in vitro observations where fibroblasts cultured in collagen-based hydrogels did not durotax toward regions with higher collagen concentrations. Instead, they exhibited an adurotactic behavior, following a more random trajectory. Overall, cell’s migratory response in 3D domains depends on its phenotype, and the properties of the surrounding environment, that is, 3D cell motion is strongly dependent on the context.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Immunopeptidomic Analyses of Colorectal Cancers With and Without Microsatellite Instability

Colorectal cancer is the second leading cause of cancer death worldwide, and the incidence of this disease is expected to increase as global socioeconomic changes occur. Immune checkpoint inhibition therapy is effective in treating a minority of colorectal cancer tumors; however, microsatellite stable tumors do not respond well to this treatment. Emerging cancer immunotherapeutic strategies aim to activate a cytotoxic T cell response against tumor-specific antigens, presented exclusively at the cell surface of cancer cells. These antigens are rare and are most effectively identified with a mass spectrometry–based approach, which allows the direct sampling and sequencing of these peptides. Although the few tumor-specific antigens identified to date are derived from coding regions of the genome, recent findings indicate that a large proportion of tumor-specific antigens originate from allegedly noncoding regions. Here, we employed a novel proteogenomic approach to identify tumor antigens in a collection of colorectal cancer–derived cell lines and biopsy samples consisting of matched tumor and normal adjacent tissue. The generation of personalized cancer databases paired with mass spectrometry analyses permitted the identification of more than 30,000 unique MHC I–associated peptides. We identified 19 tumor-specific antigens in both microsatellite stable and unstable tumors, over two-thirds of which were derived from noncoding regions. Many of these peptides were derived from source genes known to be involved in colorectal cancer progression, suggesting that antigens from these genes could have therapeutic potential in a wide range of tumors. These findings could benefit the development of T cell–based vaccines, in which T cells are primed against these antigens to target and eradicate tumors. Such a vaccine could be used in tandem with existing immune checkpoint inhibition therapies, to bridge the gap in treatment efficacy across subtypes of colorectal cancer with varying prognoses. Data are available via ProteomeXchange with identifier PXD028309.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

New Insights on Effects of Glucocorticoids in Patients With SARS-CoV-2 Infection

ObjectiveSince January 2020, the highly contagious novel coronavirus SARS-CoV-2 has caused a global pandemic. Severe COVID-19 leads to a massive release of proinflammatory mediators, leading to diffuse damage to the lung parenchyma, and the development of acute respiratory distress syndrome. Treatment with the highly potent glucocorticoid (GC) dexamethasone was found to be effective in reducing mortality in severely affected patients.MethodsTo review the effects of glucocorticoids in the context of COVID-19 we performed a literature search in the PubMed database using the terms COVID-19 and glucocorticoid treatment. We identified 1429 article publications related to COVID-19 and glucocorticoid published from 1.1.2020 to the present including 238 review articles and 36 Randomized Controlled Trials. From these studies, we retrieved 13 Randomized Controlled Trials and 86 review articles that were relevant to our review topics. We focused on the recent literature dealing with glucocorticoid metabolism in critically ill patients and investigating the effects of glucocorticoid therapy on the immune system in COVID-19 patients with severe lung injury.ResultsIn our review, we have discussed the regulation of the hypothalamic-pituitary-adrenal axis in patients with critical illness, selection of a specific GC for critical illness-related GC insufficiency, and recent studies that investigated hypothalamic-pituitary-adrenal dysfunction in patients with COVID-19. We have also addressed the specific activation of the immune system with chronic endogenous glucocorticoid excess, as seen in patients with Cushing syndrome, and, finally, we have discussed immune activation due to coronavirus infection and the possible mechanisms leading to improved outcomes in patients with COVID-19 treated with GCs.ConclusionFor clinical endocrinologists prescribing GCs for their patients, a precise understanding of both the molecular- and cellular-level mechanisms of endogenous and exogenous GCs is imperative, including timing of administration, dosage, duration of treatment, and specific formulations of GCs.     

MS-based technologies for untargeted single-cell proteomics

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The Effect of Gender-Affirming Hormone Therapy on the Risk of Subclinical Atherosclerosis in the Transgender Population: A Systematic Review

ObjectiveThe impact of gender-affirming hormone therapy (GAHT) on cardiovascular (CV) health is still not entirely established. A systematic review was conducted to summarize the evidence on the risk of subclinical atherosclerosis in transgender people receiving GAHT.MethodsA systematic review was performed following Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines, and data were searched in PubMed, LILACS, EMBASE, and Scopus databases for cohort, case-control, and cross-sectional studies or randomized clinical trials, including transgender people receiving GAHT. Transgender men and women before and during/after GAHT for at least 2 months, compared with cisgender men and women or hormonally untreated transgender persons. Studies reporting changes in variables related to endothelial function, arterial stiffness, autonomic function, and blood markers of inflammation/coagulation associated with CV risk were included.ResultsFrom 159 potentially eligible studies initially identified, 12 were included in the systematic review (8 cross-sectional and 4 cohort studies). Studies of trans men receiving GAHT reported increased carotid thickness, brachial-ankle pulse wave velocity, and decreased vasodilation. Studies of trans women receiving GAHT reported decreased interleukin 6, plasminogen activator inhibitor-1, and tissue plasminogen activator levels and brachial-ankle pulse wave velocity, with variations in flow-mediated dilation and arterial stiffness depending on the type of treatment and route of administration.ConclusionsThe results suggest that GAHT is associated with an increased risk of subclinical atherosclerosis in transgender men but may have either neutral or beneficial effects in transgender women. The evidence produced is not entirely conclusive, suggesting that additional studies are warranted in the context of primary prevention of CV disease in the transgender population receiving GAHT.Systematic Review RegistrationPROSPERO, identifier CRD42022323757.     

Growth performance,haematological assessment and chemical composition of Nile tilapia,Oreochromis niloticus (Linnaeus, 1758) fed different levels of Aloe vera extract as feed additives in a closed aquaculture system

A 105-day experimental trial was conducted to assess different levels of dietary Aleo vera extract supplementation on water quality parameters, proximate composition, growth performance and haematological parameters of fry Oreochromis niloticus. Four different percentages of dietary leaf extract powder of Aleo vera (ALE) with a basal feed, designated as, i.e., T0 (Control group; without ALE), T1 (1% ALE), T2 (2% ALE), and T3 (3% ALE). Fish fry was reared in concrete tanks (7.0 m, 1.6 m, 1.0: L, W, H; water volume 11.2 m3/tank), with an average initial weight 4.04 ± 0.03 g/ fry, and each treatment was triplicated. Fry was randomly distributed at a stocking rate of 450 individuals/ tanks. The water quality parameters revealed that temperature, pH, salinity, dissolved oxygen (DO) and nitrates were found in a promising range as given by FAO/WHO limits. However, the record values obtained for Electric Conductivity (EC), Total dissolved solids (TDS), and alkalinities were not found in all tanks' suitable range according to FAO/WHO limits. The results revealed a significant impact of different percentages of dietary ALE supplementation on fry's body composition and haematological parameters. Moreover, the final body weight, final body length, average daily weight gain (g), net weight gain (g) and specific growth rate (%) were significantly higher (p < 0.05) in T1 and T2 compared with T0 and T3 treatments. The poorest feed conversion ratio was recorded in the T2 group compared with other treatments. Thus, the current study provides information about the nutritional quality of Nile tilapia culturing in Pakistan.