Discovery of antimicrobial peptides in the global microbiome with machine learning.

Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine-learning-based approach to predict antimicrobial peptides (AMPs) within the global microbiome and leverage a vast dataset of 63,410 metagenomes and 87,920 prokaryotic genomes from environmental and host-associated habitats to create the AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, few of which match existing databases. AMPSphere provides insights into the evolutionary origins of peptides, including by duplication or gene truncation of longer sequences, and we observed that AMP production varies by habitat. To validate our predictions, we synthesized and tested 100 AMPs against clinically relevant drug-resistant pathogens and human gut commensals both in vitro and in vivo. A total of 79 peptides were active, with 63 targeting pathogens. These active AMPs exhibited antibacterial activity by disrupting bacterial membranes. In conclusion, our approach identified nearly one million prokaryotic AMP sequences, an open-access resource for antibiotic discovery.

Tracing human life trajectory using gut microbial communities by context-aware deep learning.

The gut microbial communities are highly plastic throughout life, and the human gut microbial communities show spatial-temporal dynamic patterns at different life stages. However, the underlying association between gut microbial communities and time-related factors remains unclear. The lack of context-awareness, insufficient data, and the existence of batch effect are the three major issues, making the life trajection of the host based on gut microbial communities problematic. Here, we used a novel computational approach (microDELTA, microbial-based deep life trajectory) to track longitudinal human gut microbial communities' alterations, which employs transfer learning for context-aware mining of gut microbial community dynamics at different life stages. Using an infant cohort, we demonstrated that microDELTA outperformed Neural Network for accurately predicting the age of infant with different delivery mode, especially for newborn infants of vaginal delivery with the area under the receiver operating characteristic curve of microDELTA and Neural Network at 0.811 and 0.436, respectively. In this context, we have discovered the influence of delivery mode on infant gut microbial communities. Along the human lifespan, we also applied microDELTA to a Chinese traveler cohort, a Hadza hunter-gatherer cohort and an elderly cohort. Results revealed the association between long-term dietary shifts during travel and adult gut microbial communities, the seasonal cycling of gut microbial communities for the Hadza hunter-gatherers, and the distinctive microbial pattern of elderly gut microbial communities. In summary, microDELTA can largely solve the issues in tracing the life trajectory of the human microbial communities and generate accurate and flexible models for a broad spectrum of microbial-based longitudinal researches.

EXPERT: transfer learning-enabled context-aware microbial community classification.

Microbial community classification enables identification of putative type and source of the microbial community, thus facilitating a better understanding of how the taxonomic and functional structure were developed and maintained. However, previous classification models required a trade-off between speed and accuracy, and faced difficulties to be customized for a variety of contexts, especially less studied contexts. Here, we introduced EXPERT based on transfer learning that enabled the classification model to be adaptable in multiple contexts, with both high efficiency and accuracy. More importantly, we demonstrated that transfer learning can facilitate microbial community classification in diverse contexts, such as classification of microbial communities for multiple diseases with limited number of samples, as well as prediction of the changes in gut microbiome across successive stages of colorectal cancer. Broadly, EXPERT enables accurate and context-aware customized microbial community classification, and potentiates novel microbial knowledge discovery.

Visible-Light-Induced Photoredox 1,2-Dialkylation of Styrenes with α-Carbonyl Alkyl Bromides and Pyridin-1-ium Salts.

A visible-light-driven photoredox dialkylation of styrenes with α-carbonyl alkyl bromides and pyridin-1-ium salts for the synthesis of polysubstituted 1,4-dihydropyridines is reported. This reaction enables the formation of two new C(sp3)-C(sp3) bonds in a single reaction step and provides a strategy that employs pyridin-1-ium salts as the functionalized alkylating reagents via dearomatization to directly trap the resulting alkyl radicals from radical addition of alkenes and then terminate the alkene dialkylation.

Nickel-Catalyzed Reductive Cross-Coupling of Propargylic Acetates with Chloro(vinyl)silanes: Access to Silylallenes.

Because of their various reactivities, propargyl acetates are refined chemical intermediates that are extensively applied in pharmaceutical synthesis. Currently, reactions between propargyl acetates and chlorosilanes may be the most effective method for synthesizing silylallenes. Nevertheless, owing to the adaptability and selectivity of substrates, transition metal catalysis is difficult to achieve. Herein, nickel-catalyzed reductive cross-coupling reactions between propargyl acetates and substituted vinyl chlorosilanes for the synthesis of tetrasubstituted silylallenes are described. Therein, metallic zinc is a crucial reductant that effectively enables two electrophilic reagents to selectively construct C(sp2)-Si bonds. Additionally, a Ni-catalyzed reductive mechanism involving a radical process is proposed on the basis of deuteration-labeled experiments.

Construction of a Decellularized Multicomponent Extracellular Matrix Interpenetrating Network Scaffold by Gelatin Microporous Hydrogel 3D Cell Culture System.

Interface tissue repair requires the construction of biomaterials with integrated structures of multiple protein types. Hydrogels that modulate internal porous structures provide a 3D microenvironment for encapsulated cells, making them promise for interface tissue repair. Currently, reduction of intrinsic immunogenicity and increase of bioactive extracellular matrix (ECM) secretion are issues to be considered in these materials. In this study, gelatin methacrylate (GelMA) hydrogel is used to encapsulate chondrocytes and construct a phase transition 3D cell culture system (PTCC) by utilizing the thermosensitivity of gelatin microspheres to create micropores within the hydrogel. The types of bioactive extracellular matrix protein formation by chondrocytes encapsulated in hydrogels are investigated in vitro. After 28 days of culture, GelMA PTCC forms an extracellular matrix predominantly composed of collagen type II, collagen type I, and fibronectin. After decellularization, the protein types and mechanical properties are well preserved, fabricating a decellularized tissue-engineered extracellular matrix and GelMA hydrogel interpenetrating network hydrogel (dECM-GelMA IPN) consisting of GelMA hydrogel as the first-level network and the ECM secreted by chondrocytes as the second-level network. This material has the potential to mediate the repair and regeneration of tendon-bone interface tissues with multiple protein types.

Evaluation of the American College of Surgeons risk calculator in hepatectomy for metastatic colorectal cancer in a Southeast Asian population.

PURPOSE: This study evaluated the accuracy of the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) calculator in predicting outcomes after hepatectomy for colorectal cancer (CRC) liver metastasis in a Southeast Asian population. METHODS: Predicted and actual outcomes were compared for 166 patients undergoing hepatectomy for CRC liver metastasis identified between 2017 and 2022, using receiver operating characteristic curves with area under the curve (AUC) and Brier score. RESULTS: The ACS-NSQIP calculator accurately predicted most postoperative complications (AUC > 0.70), except for surgical site infection (AUC = 0.678, Brier score = 0.045). It also exhibited satisfactory performance for readmission (AUC = 0.818, Brier score = 0.011), reoperation (AUC = 0.945, Brier score = 0.002), and length of stay (LOS, AUC = 0.909). The predicted LOS was close to the actual LOS (5.9 vs. 5.0 days, P = 0.985). CONCLUSION: The ACS-NSQIP calculator demonstrated generally accurate predictions for 30-day postoperative outcomes after hepatectomy for CRC liver metastasis in our patient population.

A hydrophilic polyimidazolium antibiotic targeting the membranes of Gram-negative bacteria.

OBJECTIVES: The rise of MDR Gram-negative bacteria (GNB), especially those resistant to last-resort drugs such as carbapenems and colistin, is a global health risk and calls for increased efforts to discover new antimicrobial compounds. We previously reported that polyimidazolium (PIM) compounds exhibited significant antimicrobial activity and minimal mammalian cytotoxicity. However, their mechanism of action is relatively unknown. We examined the efficacy and mechanism of action of a hydrophilic PIM (PIM5) against colistin- and meropenem-resistant clinical isolates. METHODS: MIC and time-kill testing was performed for drug-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. N-phenyl-1-naphthylamine and propidium iodide dyes were employed to determine membrane permeabilization. Spontaneous resistant mutants and single deletion mutants were generated to understand potential resistance mechanisms to the drug. RESULTS: PIM5 had the same effectiveness against colistin- and meropenem-resistant strains as susceptible strains of GNB. PIM5 exhibited a rapid bactericidal effect independent of bacterial growth phase and was especially effective in water. The polymer disrupts both the outer and cytoplasmic membranes. PIM5 binds and intercalates into bacterial genomic DNA upon entry of cells. GNB do not develop high resistance to PIM5. However, the susceptibility and uptake of the polymer is moderately affected by mutations in the two-component histidine kinase sensor BaeS. PIM5 has negligible cytotoxicity on human cells at bacterial-killing concentrations, comparable to the commercial antibiotics polymyxin B and colistin. CONCLUSIONS: PIM5 is a potent broad-spectrum antibiotic targeting GNB resistant to last-resort antibiotics.

Designer broad-spectrum polyimidazolium antibiotics.

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

Fabrication and Performance Evaluation of Gelatin/Sodium Alginate Hydrogel-Based Macrophage and MSC Cell-Encapsulated Paracrine System with Potential Application in Wound Healing.

A gelatin/sodium alginate-based hydrogel microsphere has been fabricated after reaction condition optimization. Macrophages (RAW246.7) and adipose mesenchymal stem cells (ADSC) have been subsequently encapsulated in the microsphere in order to construct a 3D paracrine system for wound healing treatment. The synthesized microsphere displayed neglectable cytotoxicity toward both encapsulated cells until 10 days of incubation, indicating promising biocompatibility of the microsphere. A qRT-PCR and ELISA experiment revealed positive regulation of cytokines (Arg-1, IL-6, IL-8, IL-10, bFGF, HGF, VEGF, TLR-1, and CXCL13) expression regarding macrophage phenotype transformation and anti-inflammatory performance both inside the microsphere and in the microenvironment of established in vitro inflammatory model. Additionally, positive tendency of cytokine expression benefit wound healing was more pronounced in a fabricated 3D paracrine system than that of a 2D paracrine system. Furthermore, the 3D paracrine system exhibited more efficiently in the wound healing rate compared to the 2D paracrine system in an in vitro model. These results suggested the current paracrine system could be potentially used as a robust wound healing dressing.

Quality and Agronomic Trait Analyses of Pyramids Composed of Wheat Genes NGli-D2, Sec-1s and 1Dx5+1Dy10.

Due to rising living standards, it is important to improve wheat's quality traits by adjusting its storage protein genes. The introduction or locus deletion of high molecular weight subunits could provide new options for improving wheat quality and food safety. In this study, digenic and trigenic wheat lines were identified, in which the 1Dx5+1Dy10 subunit, and NGli-D2 and Sec-1s genes were successfully polymerized to determine the role of gene pyramiding in wheat quality. In addition, the effects of ω-rye alkaloids during 1BL/1RS translocation on quality were eliminated by introducing and utilizing 1Dx5+1Dy10 subunits through gene pyramiding. Additionally, the content of alcohol-soluble proteins was reduced, the Glu/Gli ratio was increased and high-quality wheat lines were obtained. The sedimentation values and mixograph parameters of the gene pyramids under different genetic backgrounds were significantly increased. Among all the pyramids, the trigenic lines in Zhengmai 7698, which was the genetic background, had the highest sedimentation value. The mixograph parameters of the midline peak time (MPT), midline peak value (MPV), midline peak width (MPW), curve tail value (CTV), curve tail width (CTW), midline value at 8 min (MTxV), midline width at 8 min (MTxW) and midline integral at 8 min (MTxI) of the gene pyramids were markedly enhanced, especially in the trigenic lines. Therefore, the pyramiding processes of the 1Dx5+1Dy10, Sec-1S and NGli-D2 genes improved dough elasticity. The overall protein composition of the modified gene pyramids was better than that of the wild type. The Glu/Gli ratios of the type I digenic line and trigenic lines containing the NGli-D2 locus were higher than that of the type II digenic line without the NGli-D2 locus. The trigenic lines with Hengguan 35 as the genetic background had the highest Glu/Gli ratio among the specimens. The unextractable polymeric protein (UPP%) and Glu/Gli ratios of the type II digenic line and trigenic lines were significantly higher than those of the wild type. The UPP% of the type II digenic line was higher than that of the trigenic lines, while the Glu/Gli ratio was slightly lower than that of the trigenic lines. In addition, the celiac disease (CD) epitopes' level of the gene pyramids significantly decreased. The strategy and information reported in this study could be very useful for improving wheat processing quality and reducing wheat CD epitopes.

Up-regulated SAMD9L modulated by TLR2 and HIF-1α as a promising biomarker in tuberculosis.

The aim of this study was to identify potential biomarkers of TB in blood and determine their function in Mtb-infected macrophages. First of all, WGCNA was used to analyse 9451 genes with significant changes in TB patients' whole blood. The 220 interferon-γ-related genes were identified, and then 30 key genes were screened using Cytoscape. Then, the AUC values of key genes were calculated to further narrow the gene range. Finally, we identified 9 genes from GSE19444. ROC analysis showed that SAMD9L, among 9 genes, had a high diagnostic value (AUC = 0.925) and a differential diagnostic value (AUC>0.865). To further narrow down the range of DEGs, the top 10 hub-connecting genes were screened from monocytes (GSE19443). Finally, we obtained 4 genes (SAMD9L, GBP1, GBP5 and STAT1) by intersections of genes from monocytes and whole blood. Among them, it was found that the function of SAMD9L was unknown after data review, so this paper studied this gene. Our results showed that SAMD9L is up-regulated and suppresses cell necrosis, and might be regulated by TLR2 and HIF-1α during Mtb infection. In addition, miR-181b-5p is significantly up-regulated in the peripheral blood plasma of tuberculosis patients, which has a high diagnostic value (AUC = 0.969).

Hypoxia-regulated carbonic anhydrase IX (CAIX) protein is an independent prognostic indicator in triple negative breast cancer.

BACKGROUND: The effect of extracellular microenvironment (hypoxia and pH) has been regarded as a key hallmark in cancer progression. The study aims to investigate the effects of carbonic anhydrase IX (CAIX), a key hypoxia-inducible marker, in triple-negative breast cancer (TNBC) in correlation with clinicopathological parameters and predicting survival outcomes. METHODS: A total of 323 TNBC cases diagnosed at the Department of Anatomical Pathology, Singapore General Hospital from 2003 to 2013 were used. Immunohistochemical staining (IHC) was performed using CAIX antibody and digital mRNA quantification was performed using NanoString assays. CAIX membranous expression was correlated with clinicopathological parameters using Chi-squared test or Fisher's exact tests. Disease-free survival (DFS) and overall-survival (OS) were estimated using Kaplan-Meier analysis and compared between groups with the log-rank test. RESULTS: Forty percent of TNBCs were observed to express CAIX protein and demonstrated significant association with larger tumour size (P = 0.002), higher histological grade (P < 0.001), and significantly worse disease-free survival (DFS) and overall survival (OS) (after adjustment: HR = 2.99, 95% CI = 1.78-5.02, P < 0.001 and HR = 2.56, 95% CI = 1.41-4.65, P = 0.002, respectively). Gene ontology enrichment analysis revealed six significantly enriched cellular functions (secretion, cellular component disassembly, regulation of protein complex assembly, glycolytic process, cellular macromolecular complex assembly, positive regulation of cellular component biogenesis) associated with genes differentially expressed (CAIX, SETX, WAS, HK2, DDIT4, TUBA4α, ARL1). Three genes (WAS, SETX and DDIT4) were related to DNA repair, indicating that DNA stability may be influenced by hypoxia in TNBC. CONCLUSIONS: Our results demonstrate that CAIX appears to be a significant hypoxia-inducible molecular marker and increased CAIX protein levels are independently associated with poor survival in TNBC. Identification of CAIX-linked seven gene-signature and its relationship with enriched cellular functions further support the implication and influence of hypoxia-mediated CAIX expression in TNBC tumour microenvironment.

Fracture Resistance of Titanium and Fiber Dental Posts: A Systematic Review and Meta-Analysis.

PURPOSE: The fracture resistance of dental post systems is influenced by the material of the post. The purpose of this systematic review and meta-analysis was to assess if there is a difference in fracture resistance between prefabricated dental titanium posts and fiber posts. METHODS: An online electronic search was performed using the PubMed, Scopus, and Web of Science databases for in vitro studies published from 2010 to 2020 in English. The retrieved eligible studies that compared the fracture resistance of titanium and fiber posts on human teeth were selected. The pooled standardized mean difference (SMD) with a 95% confidence interval was calculated. In addition, the trial sequential analysis (TSA) was performed to test if the available studies are sufficient to make conclusive evidence. RESULTS: Of the 1165 retrieved studies, 17 studies were included in the qualitative analysis, while 16 studies were included in the quantitative analysis. Because of the high heterogeneity among studies, data from 10 studies were pooled and submitted to TSA. A total of 852 teeth were evaluated for fracture of the posts in 27 independent comparison groups. The pooled effect of the residual studies revealed no significant difference between titanium and fiber posts (SMD = -0.12; 95% CI = -0.30, 0.06; p = 0.20). Results of the TSA revealed no conclusive evidence. CONCLUSIONS: The results of the current evidence revealed no significant difference between fiber and titanium posts. The evidence is insufficient, and more standardized in vitro studies are required.

Janus Particle Preparation through UV-Induced Partial Photodegradation of Spin-Coated Particle Films.

Janus particles contain two or more chemical properties typically on opposing faces. With various property combinations possible, there are several potential applications, such as surfactants and drug delivery. However, scaling up the particle production process at reasonable cost is a limiting factor, and the method reported here aims to circumvent this issue. The process is based on a top-down destructive strategy that consists of two steps. Photocatalytic titanium dioxide particles prefunctionalized with a surface coating were assembled as particle films via spin-coating on a substrate. The particle films were placed directly under an ultraviolet light source, which induced the photodegradation of the surface coating only on the particle surfaces exposed to the light. The generated Janus particles were amphiphobic-amphiphilic in character. The Janus particles had a theoretical Janus balance close to ideal and remained attached at a hexane/water interface after disruption. They were able to make Pickering emulsions of water in silicone oil with a low energy input. The reported method may be easily scaled up to facilitate the production of gram-scale yields. The use of UV is clean and efficient and can be applied to semiconductor particles with surface coatings that are susceptible to photodegradation, making this method highly versatile.

BODIPY-Appended Pt(II) Complexes with High Toxicities and Anti-chemoresistance Performances in a Cisplatin Resistant In Vivo Model.

Two novel fluorophore (BODIPY)-bearing complexes, pyriplatin (mCBP) and pyrimidine-chelated cisplatin (dCBP), were synthesized and characterized. The additional BODIPY-pyridine/pyridimine motifs of the two Pt(II) complexes resulted in stronger interactions with DNA in comparison with those of cisplatin. mCBP and cisplatin caused relative decreases in life span and body length in a cisplatin resistant in vivo model, N2 (wild-type) Caenorhabditis elegans. In contrast, dCBP resulted in a dramatic reduction in the two physiological parameters in N2 C. elegans, indicating high toxicity and sensitivity. The resistance factors (RF) of cisplatin, mCBP, and dCBP were determined to be 2.46, 1.04, and 0.91, respectively. The increasing RF folds for mCBP and dCBP against cisplatin were 2.36 and 2.70, respectively. This suggested they were featured with improved anti-chemoresistance capabilities. It is noteworthy that dCBP showed lowest lethal concentration (LC50) values of 0.56 and 0.61 mM in cisplatin resistant and sensitive in vivo models, respectively. Upregulation of several evolutionary conservation genes that regulate cisplatin chemoresistance through cisplatin effluxing, the DNA damage response, the unfolded protein response, and detoxification (asna-1, parp-1, enpl-1, and skn-1) was observed upon exposure to cisplatin but not to mCBP and dCBP. This could explain the improved anti-chemoresistance performances of synthesized Pt(II) complexes.

Tertiary lymphoid structures and associated plasma cells play an important role in the biology of triple-negative breast cancers.

PURPOSE: Triple-negative breast cancers (TNBC) are aggressive tumours that exhibit abundant lymphoid infiltrates which modulate tumour behaviour. Recent findings suggest that TNBC with higher densities of plasma cells are associated with a favourable prognosis, and tertiary lymphoid structures (TLS) have prognostic significance. Here, we studied the phenotype and function of plasma cells in TNBCs by assessing their association with IgG Kappa light chain expression, B cells, and TLS. METHODS: A retrospective analysis of 269 TNBC cases was performed. Tumour-infiltrating CD38+ plasma cells, CD20+ B cells, and TLS were evaluated on conventional haematoxylin-eosin-stained and immunohistochemical-stained sections of TNBC. We then selected TNBC cases demonstrating the highest and lowest densities of plasma cells, and examined their association with TLS, B cells, as well as immunoglobulin expression using Opal-Vectra multiplex immunofluorescence (IF). RESULTS: TNBC with high density of plasma cells showed significantly higher numbers of IgG Kappa+ CD38+ cells (p = 0.0089, p < 0.0001), and higher numbers of TLS (p < 0.0001), compared to TNBC with low density of plasma cells. TNBC with high density of plasma cells also showed higher numbers of CD20+ B cells in the tumour core (p < 0.0001), invasive margin (p < 0.0001), as well as stromal (p = 0.015) compartments. CONCLUSION: TNBC with high density of plasma cells are associated with higher numbers of IgG Kappa+ CD38+ cells, CD20+ B cells, and TLS. Further studies to characterize the function of plasma cell infiltrates and how they may interact with other tumour-infiltrating lymphocytes and TLS in TNBC may help improve existing immunotherapy strategies.

Novel Antimicrobial Coating on Silicone Contact Lens Using Glycidyl Methacrylate and Polyethyleneimine Based Polymers.

Contact lenses are medical devices commonly used to correct refractive errors and to maintain ocular health. Microorganisms such as bacteria that grow on the lens surface cause irritation to the eyes and can even cause loss of vision. In this paper, two different coating strategies are employed to form an efficient antimicrobial coating on contact lenses. In the first method, a presynthesized copolymer of polyethyleneimine-graft-polyethylene glycol methacrylate (PEI-PEGMA) is used and the coated lenses show antimicrobial activity (in vitro) against methicillin-resistant Staphylococcus aureus (MRSA) bacteria with killing efficacy >99.99% and log reduction of 5.1 and proxy host cell viability of 79%. In the second method, commercially available monomers/polymers such as glycidyl methacrylate (GMA), sulfobetaine methacrylate, and polyethyleneimine are used. A typical formulation consisting of 1% GMA shows antibacterial activity against MRSA with killing efficacy >99.99% and log reduction of 6.3. Proxy host cell viability for the coated lenses is found to be 90% indicating that the coating is nontoxic. Antibacterial coating reported here is very effective in killing gram-positive bacteria such as MRSA and S. aureus. The second method using commercially available monomers/polymers involving a simple coating procedure is also easily scalable.

Lactobacillus plantarum DR7 Modulated Bowel Movement and Gut Microbiota Associated with Dopamine and Serotonin Pathways in Stressed Adults.

We have previously reported that the administration of Lactobacillus plantarum DR7 for 12 weeks reduced stress and anxiety in stressed adults as compared to the placebo group, in association with changes along the brain neurotransmitters pathways of serotonin and dopamine-norepinephrine. We now aim to evaluate the effects of DR7 on gut functions, gut microbiota compositional changes, and determine the correlations between microbiota changes and the pathways of brain neurotransmitters. The administration of DR7 prevented an increase of defecation frequency over 12 weeks as compared to the placebo (p = 0.044), modulating the increase of stress-induced bowel movement. Over 12 weeks, alpha diversity of gut microbiota was higher in DR7 than the placebo group across class (p = 0.005) and order (p = 0.018) levels, while beta diversity differed between groups at class and order levels (p < 0.001). Differences in specific bacterial groups were identified, showing consistency at different taxonomic levels that survived multiplicity correction, along the phyla of Bacteroides and Firmicutes and along the classes of Deltaproteobacteria and Actinobacteria. Bacteroidetes, Bacteroidia, and Bacteroidales which were reduced in abundance in the placebo group showed opposing correlation with gene expression of dopamine beta hydrolase (DBH, dopamine pathway; p < 0.001), while Bacteroidia and Bacteroidales showed correlation with tryptophan hydroxylase-II (TPH2, serotonin pathway; p = 0.001). A correlation was observed between DBH and Firmicutes (p = 0.002), Clostridia (p < 0.001), Clostridiales (p = 0.001), Blautia (p < 0.001), and Romboutsia (p < 0.001), which were increased in abundance in the placebo group. Blautia was also associated with TDO (p = 0.001), whereas Romboutsia had an opposing correlation with TPH2 (p < 0.001). Deltaproteobacteria and Desulfovibrionales which were decreased in abundance in the placebo group showed opposing correlation with DBH (p = 0.001), whereas Bilophila was associated with TPH2 (p = 0.001). Our present data showed that physiological changes induced by L. plantarum DR7 could be associated with changes in specific taxa of the gut microbiota along the serotonin and dopamine pathways.

Evaluation of phospho-histone H3 in Asian triple-negative breast cancer using multiplex immunofluorescence.

PURPOSE: We used multiplex immunofluorescence (mIF) to determine whether mitotic rate represents an independent prognostic marker in triple-negative breast cancer (TNBC). Secondary aims were to confirm the prognostic significance of immune cells in TNBC, and to investigate the relationship between immune cells and proliferating tumour cells. METHODS: A retrospective Asian cohort of 298 patients with TNBC diagnosed from 2003 to 2015 at the Singapore General Hospital was used in the present study. Formalin-fixed, paraffin-embedded breast cancer samples were analysed on tissue microarrays using mIF, which combined phospho-histone H3 (pHH3) expression with cytokeratin (CK) and leukocyte common antigen (CD45) expression to identify tumour and immune cells, respectively. RESULTS: Multivariate analysis showed that a high pHH3 index was associated with significantly improved overall survival (OS; p = 0.004), but this was not significantly associated with disease-free survival (DFS; p = 0.22). Similarly, multivariate analysis also revealed that a pHH3 positive count of > 1 cell per high-power field in the malignant epithelial compartment was an independent favourable prognostic marker for OS (p = 0.033) but not for DFS (p = 0.250). Furthermore, a high CD45 index was an independent favourable prognostic marker for DFS (p = 0.018), and there was a significant positive correlation between CD45 and pHH3 index (Spearman rank correlation coefficient, 0.250; p < 0.001). CONCLUSIONS: Mitotic rates as determined by pHH3 expression in epithelial cells are significantly associated with improved survival in TNBC. mIF analysis of pHH3 in combination with CK and CD45 could help clinicians in prognosticating patients with TNBC.