Tunable PhenoCycler imaging of the murine pre-clinical tumour microenvironments.

BACKGROUND: The tumour microenvironment (TME) consists of tumour-supportive immune cells, endothelial cells, and fibroblasts. PhenoCycler, a high-plex single cell spatial biology imaging platform, is used to characterize the complexity of the TME. Researchers worldwide harvest and bank tissues from mouse models which are employed to model a plethora of human disease. With the explosion of interest in spatial biology, these panoplies of archival tissues provide a valuable resource to answer new questions. Here, we describe our protocols for developing tunable PhenoCycler multiplexed imaging panels and describe our open-source data analysis pipeline. Using these protocols, we used PhenoCycler to spatially resolve the TME of 8 routinely employed pre-clinical models of lymphoma, breast cancer, and melanoma preserved as FFPE. RESULTS: Our data reveal distinct TMEs in the different cancer models that were imaged and show that cell-cell contacts differ depending on the tumour type examined. For instance, we found that the immune infiltration in a murine model of melanoma is altered in cellular organization in melanomas that become resistant to αPD-1 therapy, with depletions in a number of cell-cell interactions. CONCLUSIONS: This work presents a valuable resource study seamlessly adaptable to any field of research involving murine models. The methodology described allows researchers to address newly formed hypotheses using archival materials, bypassing the new to perform new mouse studies.

Effects of functional performance and national health insurance cost on length of hospitalization for postacute care in stroke: a retrospective observational study.

BACKGROUND: The postacute care for cerebrovascular disease (PAC-CVD) program was launched in Taiwan nearly a decade ago. However, no clear regulations regarding length of stay (LOS) in the program and extension standards exist. Thus, the allocation of limited medical resources such as hospital beds is a major issue. METHODS: This novel study retrospectively investigated the effects of functional performance and national health insurance (NHI) costs on PAC-CVD LOS. Data for 263 patients with stroke who participated in the PAC-CVD program were analysed. Hierarchical multiple regression was used to estimate the effects of functional performance and NHI costs on LOS at three time points: weeks 3, 6, and 9. RESULTS: At week 3, age, NHI costs, modified Rankin scale score, and Barthel index significantly affected LOS, whereas at week 6, age and NHI costs were significant factors. However, functional performance and NHI costs were not significant factors at week 9. CONCLUSIONS: The study provides crucial insights into the factors affecting LOS in the PAC-CVD program, and the results can enable medical decision-makers and health care teams to develop inpatient rehabilitation plans or provide transfer arrangements tailored to patients. Specifically, this study highlights the importance of early functional recovery and consideration of NHI costs when managing LOS in the PAC-CVD program.

Engineering acetylation platform for the total biosynthesis of D-amino acids.

Optically pure D-amino acids are key chemicals with various applications. Although the production of specific D-amino acids has been achieved by chemical synthesis or with in vitro enzyme catalysts, it is challenging to convert a simple carbon source into D-amino acids with high efficiency. Here, we design an artificial metabolic pathway by engineering bacteria to heterologously express racemase and N-acetyltransferase to produce N-acetyl-D-amino acids from L-amino acids. This new platform allows the cytotoxicity of D-amino acids to be avoided. The universal potential of this acetylation protection strategy for effectively synthesizing optically pure D-amino acids is demonstrated by testing sixteen amino acid targets. Furthermore, we combine pathway optimization and metabolic engineering in Escherichia coli and achieve practically useful efficiency with four specific examples, including N-acetyl-D-valine, N-acetyl-D-serine, N-acetyl-D-phenylalanine and N-acetyl-D-phenylglycine, with titers reaching 5.65 g/L, 5.25 g/L, 8.025 g/L and 130 mg/L, respectively. This work opens up opportunities for synthesizing D-amino acids directly from simple carbon sources, avoiding costly and unsustainable conventional approaches.

Inhibition of the MNK1/2-eIF4E Axis Augments Palbociclib-Mediated Antitumor Activity in Melanoma and Breast Cancer.

Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E. When phosphorylated, eIF4E specifically engenders the translation of mRNAs that code for proteins involved in cell survival. We hypothesized that cancer cells treated with palbociclib use upregulated phosphorylated eIF4E (phospho-eIF4E) to escape the antitumor benefits of this drug. Indeed, we found that pharmacologic or genetic disruption of MNK1/2 activity, the only known kinases for eIF4E, enhanced the ability of palbociclib to decrease clonogenic outgrowth. Moreover, a quantitative proteomics analysis of melanoma cells treated with combined MNK1/2 and CDK4/6 inhibitors showed downregulation of proteins with critical roles in cell-cycle progression and mitosis, including AURKB, TPX2, and survivin. We also observed that palbociclib-resistant breast cancer cells have higher basal levels of phospho-eIF4E, and that treatment with MNK1/2 inhibitors sensitized these palbociclib-resistant cells to CDK4/6 inhibition. In vivo we demonstrate that the combination of MNK1/2 and CDK4/6 inhibition significantly increases the overall survival of mice compared with either monotherapy. Overall, our data support MNK1/2 inhibitors as promising drugs to potentiate the antineoplastic effects of palbociclib and overcome therapy-resistant disease.

Efficacy and Cost over 12 Hospitalization Weeks of Postacute Care for Stroke.

Few studies have investigated changes in functional outcomes and economic burden in patients in the postacute care cerebrovascular disease (PAC-CVD) program. We, for the first time, retrospectively investigated changes in functional performance and the national health insurance (NHI) cost over 12 PAC-CVD hospitalization weeks and evaluated the therapeutic effects of the PAC-CVD program on the NHI cost. Specifically, the functional outcomes and NHI cost of 263 stroke patients in the PAC-CVD program were analyzed. The repeated measures t test was used to compare functional performance over 0-3 weeks, and a one-way repeated measures multivariate analysis of variance was used to compare functional performance and NHI costs during weeks 0-6 and 0-9. The Wilcoxon signed-rank test was used to compare functional performance over weeks 9-12. Hierarchical multiple regression was used to estimate the effects of functional performance on NHI costs during weeks 3, 6, and 9. Over weeks 0-12, all functional performance measures demonstrated significant improvements. Changes in NHI costs varied depending on whether hospitalization was extended. At any time point, functional performance did not have a significant impact on NHI cost. Therefore, the PAC-CVD program may aid patients with stroke in sustainably regaining functional performance and effectively controlling economic burden.

Parkinson's Disease and the Gut Microbiome in Rural California.

BACKGROUND: Increasing evidence connects the gut microbiome to Parkinson's disease (PD) etiology, but little is known about microbial contributions to PD progression and its clinical features. OBJECTIVE: We aim to explore the association between the gut microbiome with PD, and the microbial association with PD-specific clinical features. METHODS: In a community-based case-control study of 96 PD patients and 74 controls, microbiome data were obtained from 16S rRNA gene sequencing of fecal samples, and analyzed for microbial diversity, taxa abundance, and predicted functional pathways that differed in PD patients and controls, and their association with PD-specific features (disease duration, motor subtypes, L-DOPA daily dose, and motor function). RESULTS: PD patients' gut microbiome showed lower species diversity (p = 0.04) and were compositionally different (p = 0.002) compared to controls but had a higher abundance of three phyla (Proteobacteria, Verrucomicrobiota, Actinobacteria) and five genera (Akkermansia, Enterococcus, Hungatella, and two Ruminococcaceae) controlling for sex, race, age, and sequencing platform. Also, 35 Metacyc pathways were predicted to be differentially expressed in PD patients including biosynthesis, compound degradation/utilization/assimilation, generation of metabolites and energy, and glycan pathways. Additionally, the postural instability gait dysfunction subtype was associated with three phyla and the NAD biosynthesis pathway. PD duration was associated with the Synergistota phylum, six genera, and the aromatic compound degradation pathways. Two genera were associated with motor function. CONCLUSION: PD patients differed from controls in gut microbiome composition and its predicted metagenome. Clinical features were also associated with bacterial taxa and altered metabolic pathways of interest for PD progression.

Arsenic 3 methyltransferase (AS3MT) automethylates on cysteine residues in vitro.

Arsenic toxicity is a global concern to human health causing increased incidences of cancer, bronchopulmonary, and cardiovascular diseases. In human and mouse, inorganic arsenic (iAs) is metabolized in a series of methylation steps catalyzed by arsenic (3) methyltransferase (AS3MT), forming methylated arsenite (MAsIII), dimethylarsenite (DMAIII) and the volatile trimethylarsine (TMA). The methylation of arsenic is coordinated by four conserved cysteines proposed to participate in catalysis, namely C33, C62, C157, and C207 in mouse AS3MT. The current model consists of AS3MT methylating iAs in the presence of the cofactor S-adenosyl-L-methionine (SAM), and the formation of intramolecular disulfide bonds following the reduction of MAsV to MAsIII. In the presence of endogenous reductants, these disulfide bonds are reduced, the enzyme re-generates, and the second round of methylation ensues. Using in vitro methylation assays, we find that AS3MT undergoes an initial automethylation step in the absence of iAs. This automethylation is enhanced by glutathione (GSH) and dithiothreitol (DTT), suggesting that reduced cysteines accept methyl groups from SAM to form S-methylcysteines. Following the addition of iAs, automethylation of AS3MT is decreased. Furthermore, using a Flag-AS3MT immunoprecipitation coupled to MS/MS, we identify both C33 and C62 as acceptors of the methyl group in vivo. Site-directed mutagenesis (C to A) revealed that three of the previously described cysteines were required for AS3MT automethylation. In vitro experiments show that automethylated AS3MT can methylate iAs in the presence of SAM. Thus, we propose that automethylated may represent an active conformation of AS3MT.

Mutated RAS-associating proteins and ERK activation in relapse/refractory diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is successfully treated with combination immuno-chemotherapy, but relapse with resistant disease occurs in ~ 40% of patients. However, little is known regarding relapsed/refractory DLBCL (rrDLBCL) genetics and alternative therapies. Based on findings from other tumors, we hypothesized that RAS-MEK-ERK signaling would be upregulated in resistant tumors, potentially correlating with mutations in RAS, RAF, or associated proteins. We analyzed mutations and phospho-ERK levels in tumor samples from rrDLBCL patients. Unlike other tumor types, rrDLBCL is not mutated in any Ras or Raf family members, despite having increased expression of p-ERK. In paired biopsies comparing diagnostic and relapsed specimens, 33% of tumors gained p-ERK expression, suggesting a role in promoting survival. We did find mutations in several Ras-associating proteins, including GEFs, GAPs, and downstream effectors that could account for increased ERK activation. We further investigated mutations in one such protein, RASGRP4. In silico modeling indicated an increased interaction between H-Ras and mutant RASGRP4. In cell lines, mutant RASGRP4 increased basal p-ERK expression and lead to a growth advantage in colony forming assays when challenged with doxorubicin. Relapsed/refractory DLBCL is often associated with increased survival signals downstream of ERK, potentially corresponding with mutations in protein controlling RAS/MEK/ERK signaling.

Unsupervised mobile cognitive testing for use in preclinical Alzheimer's disease.

INTRODUCTION: Unsupervised digital cognitive testing is an appealing means to capture subtle cognitive decline in preclinical Alzheimer's disease (AD). Here, we describe development, feasibility, and validity of the Boston Remote Assessment for Neurocognitive Health (BRANCH) against in-person cognitive testing and amyloid/tau burden. METHODS: BRANCH is web-based, self-guided, and assesses memory processes vulnerable in AD. Clinically normal participants (n = 234; aged 50-89) completed BRANCH; a subset underwent in-person cognitive testing and positron emission tomography imaging. Mean accuracy across BRANCH tests (Categories, Face-Name-Occupation, Groceries, Signs) was calculated. RESULTS: BRANCH was feasible to complete on participants' own devices (primarily smartphones). Technical difficulties and invalid/unusable data were infrequent. BRANCH psychometric properties were sound, including good retest reliability. BRANCH was correlated with in-person cognitive testing (r = 0.617, P < .001). Lower BRANCH score was associated with greater amyloid (r = -0.205, P = .007) and entorhinal tau (r = -0.178, P = .026). DISCUSSION: BRANCH reliably captures meaningful cognitive information remotely, suggesting promise as a digital cognitive marker sensitive early in the AD trajectory.

Palladium/Brønsted-Acid-Catalyzed Diastereoselective Cyclization with Chiral Sulfinamides as Nucleophiles.

This article reports diastereoselective cyclization with chiral sulfinamides as nucleophiles in two reaction pathways: (1) intramolecular allylic substitution and (2) sequential aerobic oxidation with aza-Michael addition. These reactions were enabled by synergistic palladium and Brønsted acid catalysis and produced chiral isoindolines with good yields of 55-92% and high diastereoselectivities of 10:1 to >20:1 dr.

The human origin recognition complex is essential for pre-RC assembly, mitosis, and maintenance of nuclear structure.

The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2-deficient cells were also large, with decompacted heterochromatin. Some ORC2-deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.

Increased Anxiety and Depression Symptoms in Post-Acute Care Patients with Stroke during the COVID-19 Pandemic.

This study aimed to explore the quality and stability of post-acute care for patients with stroke, including their functional outcomes, mental health and medical care in Taiwan during the COVID-19 pandemic. In this retrospective case-control study-based on propensity score matching-we assessed 11 patients admitted during the pandemic period (in 2021) and 11 patients admitted during the non-pandemic period (in 2020). Functional outcomes, including the scores of the modified Rankin Scale, Barthel Index, EuroQoL-5 Dimension, Lawton-Brody instrumental activities of daily living, Berg Balance Scale, 5-metre walking speed and 6-min walking distance, were determined. Data on the length of acute care, length of post-acute care, destination after discharge and 14-days readmission were used to evaluate the quality of medical care. The Wilcoxon signed-rank test was used to compare functional performance before and after rehabilitation. The pandemic group showed no significant improvement in the scores of EuroQoL-5 Dimension, a self-reported health status assessment (p = 0.13), with the anxiety or depression dimension showing a negative effect (r = 0.21). Post-acute care programmes can efficiently improve the functional performance of patients with stroke during the COVID-19 pandemic in Taiwan. Mental health should therefore be simultaneously maintained while rehabilitating physical function.

Tungsten Increases Sex-Specific Osteoclast Differentiation in Murine Bone.

Tungsten is a naturally occurring metal that is increasingly used in industry and medical devices, and is labeled as an emerging environmental contaminant. Like many metals, tungsten accumulates in bone. Our previous data indicate that tungsten decreases differentiation of osteoblasts, bone-forming cells. Herein, we explored the impact of tungsten on osteoclast differentiation, which function in bone resorption. We observed significantly elevated osteoclast numbers in the trabecular bone of femurs following oral exposure to tungsten in male, but not female mice. In order to explore the mechanism(s) by which tungsten increases osteoclast number, we utilized in vitro murine primary and cell line pre-osteoclast models. Although tungsten did not alter the adhesion of osteoclasts to the extracellular matrix protein, vitronectin, we did observe that tungsten enhanced RANKL-induced differentiation into tartrate-resistant acid phosphatase (TRAP)-positive mononucleated osteoclasts. Importantly, tungsten alone had no effect on differentiation or on the number of multinucleated TRAP-positive osteoclasts. Enhanced RANKL-induced differentiation correlated with increased gene expression of differentiated osteoclast markers Nfatc1, Acp5, and Ctsk. Although tungsten did not alter the RANK surface receptor expression, it did modulate its downstream signaling. Co-exposure of tungsten and RANKL resulted in sustained positive p38 signaling. These findings demonstrate that tungsten enhances sex-specific osteoclast differentiation, and together with previous findings of decreased osteoblastogenesis, implicate tungsten as a modulator of bone homeostasis.

A Functional Assay to Assess Toxicity During Murine B Cell Development In Vitro.

B lymphocytes, or B cells, are important players in immunity that produce antigen-specific immunoglobulins. As a result, they are involved in various immune-linked pathologies. To better understand, prevent, or treat B cell-associated disease and immunotoxicity, we developed an in vitro assay to model early murine B cell differentiation within the bone marrow. This model uses sorted B cell precursors cultured on a supporting stromal cell layer, which over time acquire markers of further differentiated B cells, such as surface antigens and rearranged immunoglobulin light chain. Importantly, we utilized our in vitro model to validate our previous observations that xenobiotics, such as tungsten and organotins, alter B cell development in vivo. Furthermore, gene expression can be modulated in this model using retroviral transduction, making it amenable to investigating signaling pathways involved in disruption of B cell differentiation. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Assessment of early B lymphocyte differentiation in vitro Support Protocol: Isolation of murine bone marrow Alternate Protocol 1: Addition of recombinant interleukin-7 Alternate Protocol 2: Genetic manipulation via retroviral transduction.

Addressing K/L-edge overlap in elemental analysis from micro-X-ray fluorescence: bioimaging of tungsten and zinc in bone tissue using synchrotron radiation and laser ablation inductively coupled plasma mass spectrometry.

Synchrotron radiation micro-X-ray fluorescence (SR-µXRF) is a powerful elemental mapping technique that has been used to map tungsten and zinc distribution in bone tissue. However, the heterogeneity of the bone samples along with overlap of the tungsten L-edge with the zinc K-edge signals complicates SR-µXRF data analysis, introduces minor artefacts into the resulting element maps, and decreases image sensitivity and resolution. To confirm and more carefully delineate these SR-µXRF results, we have employed laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to untangle the problem created by the K/L-edge overlap of the tungsten/zinc pair. While the overall elemental distribution results are consistent between the two techniques, LA-ICP-MS provides significantly higher sensitivity and image resolution compared with SR-µXRF measurements in bone. These improvements reveal tissue-specific distribution patterns of tungsten and zinc in bone, not observed using SR-µXRF. We conclude that probing elemental distribution in bone is best achieved using LA-ICP-MS, though SR-µXRF retains the advantage of being a non-destructive method with the capability of being paired with X-ray techniques, which determine speciation in situ. Since tungsten is an emerging contaminant recently found to accumulate in bone, accurately determining its distribution and speciation in situ is essential for directing toxicological studies and informing treatment regimes. Graphical abstract Tungsten and zinc localization and uptake in mouse femurs were imaged by synchrotron radiation, left, and by laser ablation ICP-MS, right. The increased resolution of the LA-ICP-MS technique resolves the problem of the overlap in tungsten's L-edge and zinc's K-edge.

Curcumin Attenuates Adipogenesis by Inducing Preadipocyte Apoptosis and Inhibiting Adipocyte Differentiation.

Patients with metabolic syndrome are at an increased risk of developing type 2 diabetes and cardiovascular diseases. The principal risk factor for development of metabolic syndrome is obesity, defined as a state of pathological hyperplasia or/and hypertrophy of adipose tissue. The number of mature adipocytes is determined by adipocyte differentiation from preadipocytes. The purpose of the present study is to investigate the effects of curcumin on adipogenesis and the underlying mechanism. To examine cell toxicity of curcumin, 3T3-L1 preadipocytes were treated with 0-50 µM curcumin for 24, 48, or 72 h, then cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The effect of curcumin on the cell cycle was determined by flow cytometry. Curcumin-induced cell apoptosis was determined by the TUNEL assay and curcumin-induced caspase activation was measured by immunoblotting. The effect of curcumin on adipocyte differentiation was determined by measuring mitotic clonal expansion (MCE), expression of adipogenic transcription factors, and lipid accumulation. Results showed the viability of preadipocytes was significantly decreased by treatment with 30 µM curcumin, a concentration that caused apoptosis in preadipocytes, as assessed by the TUNEL assay, and caused activation of caspases 8, 9, and 3. A non-cytotoxic dose of curcumin (15 µM) inhibited MCE, downregulated the expression of PPARγ and C/EBPα, prevented differentiation medium-induced ß-catenin downregulation, and decreased the lipid accumulation in 3T3-L1 adipocytes. In conclusion, our data show that curcumin can induce preadipocyte apoptosis and inhibit adipocyte differentiation, leading to suppression of adipogenesis.

Isolation and characterization of Chlorella sp. mutants with enhanced thermo- and CO2 tolerances for CO2 sequestration and utilization of flue gases.

BACKGROUND: The increasing emission of flue gas from industrial plants contributes to environmental pollution, global warming, and climate change. Microalgae have been considered excellent biological materials for flue gas removal, particularly CO2 mitigation. However, tolerance to high temperatures is also critical for outdoor microalgal mass cultivation. Therefore, flue gas- and thermo-tolerant mutants of Chlorella vulgaris ESP-31 were generated and characterized for their ability to grow under various conditions. RESULTS: In this study, we obtained two CO2- and thermo-tolerant mutants of Chlorella vulgaris ESP-31, namely, 283 and 359, with enhanced CO2 tolerance and thermo-tolerance by using N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagenesis followed by screening at high temperature and under high CO2 conditions with the w-zipper pouch selection method. The two mutants exhibited higher photosynthetic activity and biomass productivity than that of the ESP-31 wild type. More importantly, the mutants were able to grow at high temperature (40 °C) and a high concentration of simulated flue gas (25% CO2, 80-90 ppm SO2, 90-100 ppm NO) and showed higher carbohydrate and lipid contents than did the ESP-31 wild type. CONCLUSIONS: The two thermo- and flue gas-tolerant mutants of Chlorella vulgaris ESP-31 were useful for CO2 mitigation from flue gas under heated conditions and for the production of carbohydrates and biodiesel directly using CO2 from flue gas.

Tungsten Blocks Murine B Lymphocyte Differentiation and Proliferation Through Downregulation of IL-7 Receptor/Pax5 Signaling.

Tungsten is an emerging environmental toxicant associated with several pediatric leukemia clusters, although a causal association has not been established. Our previous work demonstrated that tungsten exposure resulted in an accumulation of pre-B cells in the bone marrow, the same cell type that accumulates in pediatric acute lymphoblastic leukemia (ALL). To better understand the relevant molecular mechanisms, we performed RNA-sequencing on flow sorted pre-B cells from control and tungsten-exposed mice. Tungsten decreased the expression of multiple genes critical for B cell development, including members of the interleukin-7 receptor (IL-7R) and pre-B cell receptor signaling pathways, such as Jak1, Stat5a, Pax5, Syk, and Ikzf3. These results were confirmed in an in vitro model of B cell differentiation, where tungsten arrested differentiation at the pro-B cell stage and inhibited proliferation. These changes were associated with decreased expression of multiple genes in the IL-7R signaling pathway and decreased percentage of IL-7R, phosphorylated STAT5 double-positive cells. Supplementation with IL-7 or overexpression of Pax5, the transcription factor downstream of IL-7R, rescued the tungsten-induced differentiation block. Together, these data support the hypothesis that IL-7R/Pax5 signaling axis is critical to tungsten-mediated effects on pre-B cell development. Importantly, many of these molecules are modulated in ALL.

Exploration of microbiota targets for major depressive disorder and mood related traits.

Growing evidence suggests the link between gut microbiota and mood regulation. The current study aimed to identify microbiota targets for major depressive disorder (MDD) and mood-related traits in Taiwanese samples, while taking into account the influence of dietary patterns. We recruited 36 MDD patients and 37 healthy controls for 16S rRNA gene sequencing. We assessed nutrient content using food frequency questionnaire, and mood related phenotypes, including depressive severity, anxiety, and perceived stress. Analysis of composition of microbiomes (ANCOM) models were performed to evaluate microbiota compositions between patients and controls, while adjusted for fat intake% and sequencing platforms. We found 23 taxa (4 phyla, 7 families and 12 genera) to be associated with depression and beta diversity was differed between groups. Phylum Actinobacteria and Firmicutes were overrepresented in MDD patients. At genus level, Bifidobacterium (7%) and Blautia (8%) had relatively high abundance among MDD patients, while Prevotella (16%) had high abundance in controls. Holdemania exhibited moderate correlation with anxiety (r = 0.65) and perceived stress level (r = 0.49) mainly in MDD patients but not controls. Pathway analyses revealed that pentose phosphate and starch and sucrose metabolism processes were important pathways for depression via microbiota functions. In conclusion, our results revealed microbiota targets for depression that are independent of fat intake. It is worthwhile to conduct further studies to replicate the current findings and to integrate with biochemistry and metabolomics data to better understand the functions of identified targets.

Improvement of outdoor culture efficiency of cyanobacteria by over-expression of stress tolerance genes and its implication as bio-refinery feedstock.

This study was undertaken to increase the biomass and carbohydrate productivities of a freshwater cyanobacterium Synechococcus elongatus under hot outdoor conditions through genetic manipulation to facilitate the application of using the cyanobacterial biomass as bio-refinery feedstocks. The stress tolerance genes (hspA, osmotin) were expressed in S. elongatus to improve their growth under various environment stresses of outdoor cultivation. The results revealed that over-expression of hspA and osmotin significantly improved temperature (45°C), high light intensity, and salt tolerances of S. elongatus cells, making it capable of efficiently growing in seawater under outdoor cultivation. The carbohydrate productivity of these stress tolerant strains was also 15-30-fold higher than that of the control strain, although the carbohydrate contents of the recombinant and control strains were similar. Our findings demonstrate that the genetic engineering for improved stresses tolerance in S. elongatus could facilitate the feasibility of using cyanobacteria as feedstock for bio-refinery industry.