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The pancreas is a vital organ with digestive and endocrine roles, and diseases of the pancreas affect millions of people yearly. A better understanding of the pancreas proteome and its dynamic post-translational modifications (PTMs) is necessary to engineer higher fidelity tissue analogues for use in transplantation. The extracellular matrix (ECM) has major roles in binding and signaling essential to the viability of insulin-producing islets of Langerhans. To characterize PTMs in the pancreas, native and decellularized tissues from four donors were analyzed. N-Glycosylated and phosphorylated peptides were simultaneously enriched via electrostatic repulsion-hydrophilic interaction chromatography and analyzed with mass spectrometry, maximizing PTM information from one workflow. A modified surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion was used to maximize solubility of the ECM. The analysis resulted in the confident identification of 3650 proteins, including 517 N-glycoproteins and 148 phosphoproteins. We identified 214 ECM proteins, of which 99 were N-glycosylated, 18 were phosphorylated, and 9 were found to have both modifications. Collagens, a major component of the ECM, were the most highly glycosylated of the ECM proteins and several were also heavily phosphorylated, raising the possibility of structural and thus functional changes resulting from these modifications. To our knowledge, this work represents the first characterization of PTMs in pancreatic ECM proteins. This work provides a basal profile of PTMs in the healthy human pancreatic ECM, laying the foundation for future investigations to determine disease-specific changes such as in diabetes and pancreatic cancer, and potentially helping to guide the development of tissue replacement constructs. Data are available via ProteomeXchange with identifier PXD025048.

Despite technical advances in introducing genomic deletions and modulating gene expression, direct inactivation of essential genes in mycobacteria remains difficult. In this study, we described clustered regularly interspaced short palindromic repeat interference (CRISPRi) technology to repress the expression of sepF (MSMEG_4219) based on nuclease-deficient CRISPR-associated protein 9 (Cas9) and small guide RNA (sgRNA) specific to the target sequence in Mycobacterium smegmatis. Using this CRISPRi approach, we achieved the repression of sepF by up to 98% in M. smegmatis without off-target effects. The depleted Msm_sepF strains resulted in growth and morphology changes including elongated, filamentous and branched bacterial cells, but the levels of the interacting partners ftsZ and murG were not modified in M. smegmatis. The sepF gene was proven to be an essential gene in M. smegmatis. This study provided an improved and detailed technical procedure for the application of CRISPRi technology in mycobacteria, and this approach was demonstrated to be a simple and efficient tool for regulating the expression of essential genes in M. smegmatis.

Extracellular matrix (ECM) is an important component of the pancreatic microenvironment which regulates β cell proliferation, differentiation, and insulin secretion. Protocols have recently been developed for the decellularization of the human pancreas to generate functional scaffolds and hydrogels. In this work, we characterized human pancreatic ECM composition before and after decellularization using isobaric dimethylated leucine (DiLeu) labeling for relative quantification of ECM proteins. A novel correction factor was employed in the study to eliminate the bias introduced during sample preparation. In comparison to the commonly employed sample preparation methods (urea and FASP) for proteomic analysis, a recently developed surfactant and chaotropic agent assisted sequential extraction/on pellet digestion (SCAD) protocol has provided an improved strategy for ECM protein extraction of human pancreatic ECM matrix. The quantitative proteomic results revealed the preservation of matrisome proteins while most of the cellular proteins were removed. This method was compared with a well-established label-free quantification (LFQ) approach which rendered similar expressions of different categories of proteins (collagens, ECM glycoproteins, proteoglycans, etc.). The distinct expression of ECM proteins was quantified comparing adult and fetal pancreas ECM, shedding light on the correlation between matrix composition and postnatal β cell maturation. Despite the distinct profiles of different subcategories in the native pancreas, the distribution of matrisome proteins exhibited similar trends after the decellularization process. Our method generated a large data set of matrisome proteins from a single tissue type. These results provide valuable insight into the possibilities of constructing a bioengineered pancreas. It may also facilitate better understanding of the potential roles that matrisome proteins play in postnatal β cell maturation.

Simple Summary The collagen architecture in the extracellular matrix (ECM) is highly remodeled in high grade serous ovarian cancer (HGSOC). Many of these tumors begin in the fallopian tubes (FT) before metastasizing to the ovaries and it is important to study ECM alterations in carcinogenesis. Here, we used Second Harmonic Generation (SHG) microscopy to classify changes in the collagen fiber morphology in normal FT, and precursor pure p53 signatures and serous tubal intraepithelial carcinoma (STICs) in tissues with no HGSOC. Using a machine learning approach based on image features, we were able to discriminate the tissue groups with good classification accuracy. We additionally performed mass spectrometry analysis of normal and HGSOC tissues to associate the differential expression of collagen isoforms with fiber morphology alterations. This work provides new insights into ECM remodeling in early stage HGSOC and suggests the combined use of SHG microscopy and mass spectrometry as a new diagnostic/prognostic approach. Abstract Background: The collagen architecture in high grade serous ovarian cancer (HGSOC) is highly remodeled compared to the normal ovary and the fallopian tubes (FT). We previously used Second Harmonic Generation (SHG) microscopy and machine learning to classify the changes in collagen fiber morphology occurring in serous tubal intraepithelial carcinoma (STIC) lesions that are concurrent with HGSOC. We now extend these studies to examine collagen remodeling in pure p53 signatures, STICs and normal regions in tissues that have no concurrent HGSOC. This is an important distinction as high-grade disease can result in distant collagen changes through a field effect mechanism. Methods: We trained a linear discriminant model based on SHG texture and image features as a classifier to discriminate the tissue groups. We additionally performed mass spectrometry analysis of normal and HGSOC tissues to associate the differential expression of collagen isoforms with collagen fiber morphology alterations. Results: We quantified the differences in the collagen architecture between normal tissue and the precursors with good classification accuracy. Through proteomic analysis, we identified the downregulation of single α-chains including those for Col I and III, where these results are consistent with our previous SHG-based supramolecular analyses. Conclusion: This work provides new insights into ECM remodeling in early ovarian cancer and suggests the combined use of SHG microscopy and mass spectrometry as a new diagnostic/prognostic approach.

The role of Sidt2 in the process of glucose and lipid metabolism has been recently reported. However, whether Sidt2 is involved in the metabolic regulation in skeletal muscle remains unknown. In this study, for the first time, using skeletal muscle-selective Sidt2 knockout mice, we found that Sidt2 was vital for the quality control of mitochondria in mouse skeletal muscle. These mice showed significantly reduced muscle tolerance and structurally abnormal mitochondria. Deletion of the Sidt2 gene resulted in decreased expression of mitochondrial fusion protein 2 (Mfn2) and Dynamin-related protein 1 (Drp1), as well as peroxisome proliferator-activated receptor γ coactivator-1 (PGC1-α). In addition, the clearance of damaged mitochondria in skeletal muscle was inhibited upon Sidt2 deletion, which was caused by blockade of autophagy flow. Mechanistically, the fusion of autophagosomes and lysosomes was compromised in Sidt2 knockout skeletal muscle cells. In summary, the deletion of the Sidt2 gene not only interfered with the quality control of mitochondria, but also inhibited the clearance of mitochondria and caused the accumulation of a large number of damaged mitochondria, ultimately leading to the abnormal structure and function of skeletal muscle.

The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation., The pancreatic extracellular matrix (ECM) is known to differ between species, age groups and physiological states, but its compositional changes throughout human life are not well understood. Here, the authors study how the proteome of pancreatic ECM changes during human development and maturation.

The lack of efficacious vaccines against Mycobacterium tuberculosis (MTB) infection is a limiting factor in the prevention and control of tuberculosis (TB), the leading cause of death from an infectious agent. Improvement or replacement of the BCG vaccine with one that reliably protects all age groups is urgent., The lack of efficacious vaccines against Mycobacterium tuberculosis (MTB) infection is a limiting factor in the prevention and control of tuberculosis (TB), the leading cause of death from an infectious agent. Improvement or replacement of the BCG vaccine with one that reliably protects all age groups is urgent. Concerns exist that antigens currently being evaluated are too homogeneous. To identify new protective antigens, we screened 1,781 proteins from a high-throughput proteome-wide protein purification study for antigenic activity. Forty-nine antigens (34 previously unreported) induced antigen-specific gamma interferon (IFN-γ) release from peripheral blood mononuclear cells (PBMCs) derived from 4,452 TB and suspected TB patients and 167 healthy donors. Three (Rv1485, Rv1705c, and Rv1802) of the 20 antigens evaluated in a BALB/c mouse challenge model showed protective efficacy, reducing lung CFU counts by 66.2%, 75.8%, and 60%, respectively. Evaluation of IgG2a/IgG1 ratios and cytokine release indicated that Rv1485 and Rv1705c induce a protective Th1 immune response. Epitope analysis of PE/PPE protein Rv1705c, the strongest candidate, identified a dominant epitope in its extreme N-terminal domain accounting for 90% of its immune response. Systematic preclinical assessment of antigens Rv1485 and Rv1705c is warranted.

Immune checkpoint inhibitors (ICIs) have ushered in a new era of cancer therapy; however, ICIs are only effective in selective patients. The efficacy of ICIs is closely related to the tumor microenvironment. Fever for a long time was thought to directly regulate the immune response, and artificial “fever” from hyperthermia modulates the tumor immune microenvironment by providing danger signals with heat shock proteins (HSPs) as well as subsequent activation of immune systems. Encouraging results have been achieved in preclinical studies focused on potential synergetic effects by combining hyperthermia with ICIs. In this review, we summarized a cluster of immune-related factors that not only make hyperthermia a treatment capable of defending against cancer but also make hyperthermia a reliable treatment that creates a type I-like tumor microenvironment (overexpression of PD-L1 and enrichment of tumor infiltrating lymphocytes) in complementary for the enhancement of the ICIs. Then we reviewed recent preclinical data of the combination regimens involving hyperthermia and ICIs that demonstrated the combined efficacy and illustrated possible approaches to further boost the effectiveness of this combination.

The Xin'an Medical School began in the Jin Dynasty (266–420), developed in the Song Dynasty (960–1279), prospered in the Ming and Qing dynasties (1368–1911), and has been passed down to the modern era. As a school of medicine with distinct regional characteristics, it has contributed to the development of traditional Chinese medicine and exerted far-reaching influence, mainly in literature resources, medical theory, clinical application, and spiritual culture. This paper intends to discuss its academic characteristics and contribution to the development of traditional Chinese medicine, focusing on its formation, academic inheritance and innovation, overseas popularization, and integration of Confucianism, Taoism, and Buddhism in medicine. Keywords: Xin'an Medical School, Regional medical schools, Ancient Huizhou, Achievements and characteristics

Objective MicroRNAs (miRNAs) have been demonstrated to be differently expressed in colorectal cancer (CRC) and were identified as biomarkers and therapeutic targets for CRC. We aimed to identify the effect of microRNA-424 (miR-424) on process of CRC. Methods Exosomes were obtained from bone marrow mesenchymal stem cells (BMSCs). MiR-424, transforming growth factor-β receptor 3 (TGFBR3) vimentin, S100A4, p-Smad1 expression in tissues and cells was measured. After treated with miR-424 inhibitor or TGFBR3 overexpression plasmid, the migration, invasion, cell cycle distribution and apoptosis of Lovo cells and exosomes-transfected Lovo cells were determined. The subcutaneous tumor models were established and the tumor growth was observed. The target relation between miR-424 and TGFBR3 was confirmed. Results MiR-424 was upregulated while TGFBR3 was downregulated in CRC tissues. TGFBR3 was targeted by miR-424. Inhibited miR-424 or elevated TGFBR3 upregulated p-Smad1, indicating that TGFBR3 mediated the Smad1 pathway, thus regulating CRC progression. MiR-424 inhibition or TGFBR3 restoration also suppressed migration and invasion of CRC cells, arrested the CRC cells at G0/G1 phase, and promoted CRC cell apoptosis. Moreover, exosomal miR-424 from BMSCs promoted CRC development. Conclusion Inhibited exosomal miR-424 from BMSCs inhibited malignant behaviors of CRC cells by targeting TGFBR3, thus suppressing the progression of CRC.

Background Tuberculous meningitis (TBM) is the most severe and frequent form of central nervous system tuberculosis. The current lack of efficient diagnostic tests makes it difficult to differentiate TBM from other common types of meningitis, especially viral meningitis (VM). Metabolomics is an important tool to identify disease-specific biomarkers. However, little metabolomic information is available on adult TBM. Methods We used 1H nuclear magnetic resonance-based metabolomics to investigate the metabolic features of the CSF from 18 TBM and 20 VM patients. Principal component analysis and orthogonal signal correction-partial least squares-discriminant analysis (OSC-PLS-DA) were applied to analyze profiling data. Metabolites were identified using the Human Metabolome Database and pathway analysis was performed with MetaboAnalyst 3.0. Results The OSC-PLS-DA model could distinguish TBM from VM with high reliability. A total of 25 key metabolites that contributed to their discrimination were identified, including some, such as betaine and cyclohexane, rarely reported before in TBM. Pathway analysis indicated that amino acid and energy metabolism was significantly different in the CSF of TBM compared with VM. Conclusions Twenty-five key metabolites identified in our study may be potential biomarkers for TBM differential diagnosis and are worthy of further investigation.

The decrease of pH level in the water affects animals living in aquatic habitat, such as crustaceans. The molecular mechanisms enabling these animals to survive this environmental stress remain unknown. To understand the modulatory function of neuropeptides in crustaceans when encountering drops in pH level, we developed and implemented a multifaceted mass spectrometric platform to investigate the global neuropeptide changes in response to water acidification in the Atlantic blue crab, Callinectes sapidus. Neural tissues were collected at different incubation periods to monitor dynamic changes of neuropeptides under different stress conditions occurring in the animal. Neuropeptide families were found to exhibit distinct expression patterns in different tissues and even each isoform had its specific response to the stress. Circulating fluid in the crabs (hemolymph) was also analyzed after 2-hour exposure to acidification condition, and together with results from tissue analysis, enabled the discovery of neuropeptides participating in the stress accommodation process as putative hormones. Two novel peptide sequences were detected in the hemolymph that appeared to be involved in the stress-related regulation in the crabs. The data have been deposited to the ProteomeXchange with identifier PXD013536.

Tuberculosis (TB) is still a serious threat to human health which is caused by mycobacterium tuberculosis (Mtb). The main reason for failure to eliminate TB is lack of clearly understanding the molecular mechanism of Mtb pathogenesis. Determining Mtb interacted proteins in human enables us to characterize the mechanism and identify potential molecular targets for TB diagnosis and treatment. However, experimantally systematic Mtb interactors are not readily available. In this study, we performed an unbiased, comprehensive two-way proteome microarray based approach to systematically screen global Mtb interactors in human and determine their binding partners of Mtb effectors. Our results, for the first time, screened 84 potential Mtb interactors in human. Bioinformatic analysis further highlighted these protein candidates might engage in a wide range of cellular functions such as activation of DNA endogenous promoters, transcription of DNA/RNA and necrosis, as well as immune-related signaling pathways. Then, using Mtb proteome microarray followed His tagged pull-down assay and Co-IP, we identified one interacting partner (Rv0577) for the protein candidate NRF1 and three binding partners (Rv0577, Rv2117, Rv2423) for SMAD2, respectively. This study gives new insights into the profile of global Mtb interactors potentially involving in Mtb pathogenesis and demonstrates a powerful strategy in the discovery of Mtb effectors.

Deep-sequencing of bacterial transcriptomes using RNA-Seq technology has made it possible to identify small non-coding RNAs, RNA molecules which regulate gene expression in response to changing environments, on a genome-wide scale in an ever-increasing range of prokaryotes. However, a simple and reliable automated method for identifying sRNA candidates in these large datasets is lacking. Here, after generating a transcriptome from an exponential phase culture of Mycobacterium tuberculosis H37Rv, we developed and validated an automated method for the genome-wide identification of sRNA candidate-containing regions within RNA-Seq datasets based on the analysis of the characteristics of reads coverage maps. We identified 192 novel candidate sRNA-encoding regions in intergenic regions and 664 RNA transcripts transcribed from regions antisense (as) to open reading frames (ORF), which bear the characteristics of asRNAs, and validated 28 of these novel sRNA-encoding regions by northern blotting. Our work has not only provided a simple automated method for genome-wide identification of candidate sRNA-encoding regions in RNA-Seq data, but has also uncovered many novel candidate sRNA-encoding regions in M. tuberculosis, reinforcing the view that the control of gene expression in bacteria is more complex than previously anticipated.

Hair cells are specialized mechanosensitive cells responsible for mediating balance and hearing within the inner ear. In mammals, hair cells are limited in number and do not regenerate. Human pluripotent stem cells (hPSCs) provide a valuable source for deriving human hair cells to study their development and design therapies to treat and/or prevent their degeneration. In this study we used a dynamic 3D Rotary Cell Culture System (RCCS) for deriving inner ear organoids from hPSCs. We show RCCS-derived organoids recapitulate stages of inner ear development and give rise to an enriched population of hair cells displaying vestibular-like morphological and physiological phenotypes, which resemble developing human fetal inner ear hair cells as well as the presence of accessory otoconia-like structures. These results show that hPSC-derived organoids can generate complex inner ear structural features and be a resource to study inner ear development.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III-A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA-seq approaches, we show it is active in invader defense and has features atypical of type III-A systems: mature CRISPR RNA (crRNA) in its crRNA-CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3'-end processing after Cas6 cleavage of repeat RNA 8 nt from its 3' end. crRNAs generated resemble mature crRNA in type I systems, having both 5' (8 nt) and 3' (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3' hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis, providing a foundation for investigating the potential of a type III-A-based genome editing system.-Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.-E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.

Anchorage of orthopedic implants depends on the interfacial bonding between the implant and the host bone as well as on the mass and microstructure of peri-implant bone, with all these factors being continuously regulated by the biological process of bone (re)modeling. In osteoporotic bone implant integration may be jeopardized not only by lower peri-implant bone quality but also by reduced intrinsic regeneration ability. The first aim of this review is to provide a critical overview of the influence of osteoporosis on bone regeneration post-implantation. Mechanical stimulation can trigger bone formation and inhibit bone resorption; thus, judicious administration of mechanical loading can be used as an effective non-pharmacological treatment to enhance implant anchorage. Our second aim is to report recent achievements on the application of external mechanical stimulation to improve the quantity of peri-implant bone. The review focuses on peri-implant bone changes in osteoporotic conditions and following mechanical loading, prevalently using small animals and in vivo monitoring approaches. We intend to demonstrate the necessity to reveal new biological information on peri-implant bone mechanobiology to better target implant anchorage and fracture fixation in osteoporotic conditions. This article is protected by copyright. All rights reserved

Stringent control of the type I interferon signaling pathways is critical to effective host immune responses, however, the molecular mechanisms that negatively regulate these pathways are still poorly understood. Here, we show that apoptosis speck-like protein (ASC), an adaptor protein of inflammasome complex, can inhibit IFN-β signaling response by interacting with mitochondrial antiviral signaling protein (MAVS). Importantly, ASC-specific siRNA knockdown enhanced virus-induced type I interferon production, with consequent reduction of virus replication. Taken together, these results suggest that ASC, as a negative regulator of the MAVS-mediated innate immunity, may play an important role in host protection upon virus infection.

Tuberculosis (TB) has exceeded HIV as the most lethal infectious disease globally for two consecutive years. Moreover, one third of the world’s population is estimated to have latent tuberculosis infection (LTBI). This is mainly because of difficulties associated with diagnosis and treatment for both TB and LTBI patients. Exosomes provide a promising research tool for TB diagnosis and treatment because they are released from various cells containing valuable biochemical information related to disease. In this study, we performed RNA-sequencing analysis on exosomes derived from clinical specimens of healthy controls (HC), active tuberculosis (ATB) and LTBI patients. Our results revealed the distinct gene expression profiles of the exosomes from LTBI and ATB patients. 1) We identified many distinct up-regulated and down-regulated differentially expressed genes (DEGs) in LTBI and ATB samples, and further screened the top-20 DEGs which might provide a potential panel for differentiation of HC, LTBI, and ATB. 2) We classified all the DEGs into six expression patterns, screened the top-20 genes in each pattern, and mainly focused on those highly expressed in LTBI and ATB. 3) Some Mycobacterium tuberculosis (Mtb) RNAs were only enriched in the exosomes of LTBI samples. 4) Pathway and function analysis further indicated an increase in deteriorating healthy signals in LTBI and ATB samples, including down-regulated signaling pathways/immune response and up-regulated apoptosis/necrosis. Our findings indicate the selective packaging of RNA cargoes into exosomes under different stages of Mtb infection, while facilitating the development of potential targets for the diagnosis, prevention and treatment of tuberculosis.