Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

Lymph-derived chemokines direct early neutrophil infiltration in the lymph nodes upon Staphylococcus aureus skin infection.

A large number of neutrophils infiltrate the lymph node (LN) within 4 h after Staphylococcus aureus skin infection (4 h postinfection [hpi]) and prevent systemic S. aureus dissemination. It is not clear how infection in the skin can remotely and effectively recruit neutrophils to the LN. Here, we found that lymphatic vessel occlusion substantially reduced neutrophil recruitment to the LN. Lymphatic vessels effectively transported bacteria and proinflammatory chemokines (i.e., Chemokine [C-X-C motif] motif 1 [CXCL1] and CXCL2) to the LN. However, in the absence of lymph flow, S. aureus alone in the LN was insufficient to recruit neutrophils to the LN at 4 hpi. Instead, lymph flow facilitated the earliest neutrophil recruitment to the LN by delivering chemokines (i.e., CXCL1, CXCL2) from the site of infection. Lymphatic dysfunction is often found during inflammation. During oxazolone (OX)-induced skin inflammation, CXCL1/2 in the LN was reduced after infection. The interrupted LN conduits further disrupted the flow of lymph and impeded its communication with high endothelial venules (HEVs), resulting in impaired neutrophil migration. The impaired neutrophil interaction with bacteria contributed to persistent infection in the LN. Our studies showed that both the flow of lymph from lymphatic vessels to the LN and the distribution of lymph in the LN are critical to ensure optimal neutrophil migration and timely innate immune protection in S. aureus infection.

Matrix Metalloproteinases: From Molecular Mechanisms to Physiology, Pathophysiology, and Pharmacology.

The first matrix metalloproteinase (MMP) was discovered in 1962 from the tail of a tadpole by its ability to degrade collagen. As their name suggests, matrix metalloproteinases are proteases capable of remodeling the extracellular matrix. More recently, MMPs have been demonstrated to play numerous additional biologic roles in cell signaling, immune regulation, and transcriptional control, all of which are unrelated to the degradation of the extracellular matrix. In this review, we will present milestones and major discoveries of MMP research, including various clinical trials for the use of MMP inhibitors. We will discuss the reasons behind the failures of most MMP inhibitors for the treatment of cancer and inflammatory diseases. There are still misconceptions about the pathophysiological roles of MMPs and the best strategies to inhibit their detrimental functions. This review aims to discuss MMPs in preclinical models and human pathologies. We will discuss new biochemical tools to track their proteolytic activity in vivo and ex vivo, in addition to future pharmacological alternatives to inhibit their detrimental functions in diseases. SIGNIFICANCE STATEMENT: Matrix metalloproteinases (MMPs) have been implicated in most inflammatory, autoimmune, cancers, and pathogen-mediated diseases. Initially overlooked, MMP contributions can be both beneficial and detrimental in disease progression and resolution. Thousands of MMP substrates have been suggested, and a few hundred have been validated. After more than 60 years of MMP research, there remain intriguing enigmas to solve regarding their biological functions in diseases.

Hox-driven conditional immortalization of myeloid and lymphoid progenitors: Uses, advantages, and future potential.

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage-like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation-a signature cellular process of the macrophage-many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation-competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage-like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow-derived macrophages. To supplement the existing data, mass spectrometry-based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow-derived macrophages and the macrophage-like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.

Genomic analyses of ciprofloxacin-resistant Neisseria gonorrhoeae isolates recovered from the largest South American metropolitan area.

We sequenced 13 Neisseria gonorrhoeae isolates exhibiting distinct susceptibility profiles and which were recovered over 12 years in the metropolitan region of São Paulo, Brazil. Whole Genome Sequencing (WGS) was performed on an Illumina MiSeq™ 2 × 300 bp paired-end reads. Bioinformatics analyses were carried out using CGE, PATRIC, and BLAST databases for manual curation of obtained genomes. Multilocus sequence typing (MLST) analysis identified seven STs, namely ST1580, ST1590, ST1901, ST1902, ST8161, ST9363, and ST15640. Moreover, a diversity of mutations was observed in MtrR/G45D-A39T, PIB/G120K-A121S, and PBP1/L421P. Mutations associated with sulfonamides (DHPS/R228S) and rifampicin (RNAP/H552N) were also detected, as well as tetracycline resistance determinants, namely rpsJ/V57M and tet(M). The results presented herein can contribute to the knowledge of N. gonorrhoeae strains circulating in Sao Paulo, Brazil.

Genomic characterization of a novel SARS-CoV-2 lineage from Rio de Janeiro, Brazil.

Almost simultaneously, several studies reported the emergence of novel SARS-CoV-2 lineages characterized by their phylogenetic and genetic distinction (1), (2), (3), (4).….

Comparison of the functional and structural characteristics of rare TSC2 variants with clinical and genetic findings.

The TSC1 and TSC2 gene products interact to form the tuberous sclerosis complex (TSC), an important negative regulator of the mechanistic target of rapamycin complex 1 (TORC1). Inactivating mutations in TSC1 or TSC2 cause TSC, and the identification of a pathogenic TSC1 or TSC2 variant helps establish a diagnosis of TSC. However, it is not always clear whether TSC1 and TSC2 variants are inactivating. To determine whether TSC1 and TSC2 variants of uncertain clinical significance affect TSC complex function and cause TSC, in vitro assays of TORC1 activity can be employed. Here we combine genetic, functional, and structural approaches to try and classify a series of 15 TSC2 VUS. We investigated the effects of the variants on the formation of the TSC complex, on TORC1 activity and on TSC2 pre-mRNA splicing. In 13 cases (87%), the functional data supported the hypothesis that the identified TSC2 variant caused TSC. Our results illustrate the benefits and limitations of functional testing for TSC.

N-Terminomics/TAILS Profiling of Macrophages after Chemical Inhibition of Legumain.

Legumain (asparaginyl endopeptidase) is the only protease with a preference for cleavage after asparagine residues. Increased legumain activity is a hallmark of inflammation, neurodegenerative diseases, and cancer, and legumain inhibitors have exhibited therapeutic effects in mouse models of these pathologies. Improved knowledge of its substrates and cellular functions is a requisite to further validation of legumain as a drug target. We, therefore, aimed to investigate the effects of legumain inhibition in macrophages using an unbiased and systematic approach. By shotgun proteomics, we identified 16 094 unique peptides in RAW264.7 cells. Among these, 326 unique peptides were upregulated in response to legumain inhibition, while 241 were downregulated. Many of these proteins were associated with mitochondria and metabolism, especially iron metabolism, indicating that legumain may have a previously unknown impact on related processes. Furthermore, we used N-terminomics/TAILS (terminal amine isotopic labeling of substrates) to identify potential substrates of legumain. We identified three new proteins that are cleaved after asparagine residues, which may reflect legumain-dependent cleavage. We confirmed that frataxin, a mitochondrial protein associated with the formation of iron-sulfur clusters, can be cleaved by legumain. This further asserts a potential contribution of legumain to mitochondrial function and iron metabolism. Lastly, we also identified a potential new cleavage site within legumain itself that may give rise to a 25 kDa form of legumain that has previously been observed in multiple cell and tissue types. Collectively, these data shed new light on the potential functions of legumain and will be critical for understanding its contribution to disease.

Characterization of BKC-1 class A carbapenemase from Klebsiella pneumoniae clinical isolates in Brazil.

Three Klebsiella pneumoniae clinical isolates demonstrating carbapenem resistance were recovered from different patients hospitalized at two medical centers in São Paulo, Brazil. Resistance to all ß-lactams, quinolones, and some aminoglycosides was observed for these isolates that were susceptible to polymyxin B. Carbapenem hydrolysis, which was inhibited by clavulanic acid, was observed for all K. pneumoniae isolates that belonged to the same pulsed-field gel electrophoresis (PFGE) type and a novel sequence type (ST), ST1781 (clonal complex 442 [CC442]). A 10-kb nonconjugative incompatibility group Q (IncQ) plasmid, denominated p60136, was transferred to Escherichia coli strain TOP10 cells by electroporation. The full sequencing of p60136 showed that it was composed of a mobilization system, ISKpn23, the phosphotransferase aph3A-VI, and a 941-bp open reading frame (ORF) that codified a 313-amino acid protein. This ORF was named bla BKC-1. Brazilian Klebsiella carbapenemase-1 (BKC-1) showed a pI of 6.0 and possessed the highest identity (63%) with a ß-lactamase of Sinorhizobium meliloti, an environmental bacterium. Hydrolysis studies demonstrated that purified BKC-1 not only hydrolyzed carbapenems but also penicillins, cephalosporins, and monobactams. However, the carbapenems were less efficiently hydrolyzed due to their very low kcat values (0.0016 to 0.031 s(-1)). In fact, oxacillin was the best substrate for BKC-1 (kcat /Km , 53,522.6 mM(-1) s(-1)). Here, we report a new class A carbapenemase, confirming the diversity and rapid evolution of ß-lactamases in K. pneumoniae clinical isolates.

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.

Laminin database: a tool to retrieve high-throughput and curated data for studies on laminins.

The Laminin(LM)-database, hosted at http://www.lm.lncc.br, is the first database focusing a non-collagenous extracellular matrix protein family, the LMs. Part of the knowledge available in this website is automatically retrieved, whereas a significant amount of information is curated and annotated, thus placing LM-database beyond a simple repository of data. In its home page, an overview of the rationale for the database is seen and readers can access a tutorial to facilitate navigation in the website, which in turn is presented with tabs subdivided into LMs, receptors, extracellular binding and other related proteins. Each tab opens into a given LM or LM-related molecule, where the reader finds a series of further tabs for 'protein', 'gene structure', 'gene expression' and 'tissue distribution' and 'therapy'. Data are separated as a function of species, comprising Homo sapiens, Mus musculus and Rattus novergicus. Furthermore, there is specific tab displaying the LM nomenclatures. In another tab, a direct link to PubMed, which can be then consulted in a specific way, in terms of the biological functions of each molecule, knockout animals and genetic diseases, immune response and lymphomas/leukemias. LM-database will hopefully be a relevant tool for retrieving information concerning LMs in health and disease, particularly regarding the hemopoietic system.

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual.

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

Tryptase ß regulation of joint lubrication and inflammation via proteoglycan-4 in osteoarthritis.

PRG4 is an extracellular matrix protein that maintains homeostasis through its boundary lubricating and anti-inflammatory properties. Altered expression and function of PRG4 have been associated with joint inflammatory diseases, including osteoarthritis. Here we show that mast cell tryptase ß cleaves PRG4 in a dose- and time-dependent manner, which was confirmed by silver stain gel electrophoresis and mass spectrometry. Tryptase-treated PRG4 results in a reduction of lubrication. Compared to full-length, cleaved PRG4 further activates NF-κB expression in cells overexpressing TLR2, -4, and -5. In the destabilization of the medial meniscus model of osteoarthritis in rat, tryptase ß and PRG4 colocalize at the site of injury in knee cartilage and is associated with disease severity. When human primary synovial fibroblasts from male osteoarthritis patients or male healthy subjects treated with tryptase ß and/or PRG4 are subjected to a quantitative shotgun proteomics and proteome changes are characterized, it further supports the role of NF-κB activation. Here we show that tryptase ß as a modulator of joint lubrication in osteoarthritis via the cleavage of PRG4.

Cystatin C is glucocorticoid responsive, directs recruitment of Trem2+ macrophages, and predicts failure of cancer immunotherapy.

Cystatin C (CyC), a secreted cysteine protease inhibitor, has unclear biological functions. Many patients exhibit elevated plasma CyC levels, particularly during glucocorticoid (GC) treatment. This study links GCs with CyC's systemic regulation by utilizing genome-wide association and structural equation modeling to determine CyC production genetics in the UK Biobank. Both CyC production and a polygenic score (PGS) capturing predisposition to CyC production were associated with increased all-cause and cancer-specific mortality. We found that the GC receptor directly targets CyC, leading to GC-responsive CyC secretion in macrophages and cancer cells. CyC-knockout tumors displayed significantly reduced growth and diminished recruitment of TREM2+ macrophages, which have been connected to cancer immunotherapy failure. Furthermore, the CyC-production PGS predicted checkpoint immunotherapy failure in 685 patients with metastatic cancer from combined clinical trial cohorts. In conclusion, CyC may act as a GC effector pathway via TREM2+ macrophage recruitment and may be a potential target for combination cancer immunotherapy.

The Omicron Lineages BA.1 and BA.2 (Betacoronavirus SARS-CoV-2) Have Repeatedly Entered Brazil through a Single Dispersal Hub.

Brazil currently ranks second in absolute deaths by COVID-19, even though most of its population has completed the vaccination protocol. With the introduction of Omicron in late 2021, the number of COVID-19 cases soared once again in the country. We investigated in this work how lineages BA.1 and BA.2 entered and spread in the country by sequencing 2173 new SARS-CoV-2 genomes collected between October 2021 and April 2022 and analyzing them in addition to more than 18,000 publicly available sequences with phylodynamic methods. We registered that Omicron was present in Brazil as early as 16 November 2021 and by January 2022 was already more than 99% of samples. More importantly, we detected that Omicron has been mostly imported through the state of São Paulo, which in turn dispersed the lineages to other states and regions of Brazil. This knowledge can be used to implement more efficient non-pharmaceutical interventions against the introduction of new SARS-CoV variants focused on surveillance of airports and ground transportation.

Kinetics Analysis of ß-Lactams Hydrolysis by OXA-50 Variants of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunist pathogen usually associated with life threatening infections and exhibits a set of intrinsic and acquired antimicrobial mechanisms. Although resistance to penicillins-like compounds is commonly associated with the chromosomal Pseudomonas-derived cephalosporinases ß-lactamase, the real contribution of OXA-50, a second chromosomally encoded ß-lactamase, remains unclear. In this study, we characterized the biochemical properties of OXA-50, OXA-488, and OXA-494. Both oxacilinases differ from OXA-50 in two amino acids each. The blaOXA-50, blaOXA-488, and blaOXA-494 were cloned into pET26b+ that was transformed into Escherichia coli DH5α strain, expressed in E. coli BL21 strain, and then purified for obtaining the hydrolytic parameters. Benzylpenicillin was the preferential substrate instead of oxacillin. Besides, OXA-488 showed a threefold increase in catalytic efficiency for benzylpenicillin, and it was twofold more efficient in hydrolyzing imipenem, compared with OXA-50, although such carbapenemase activity was considered weak. In addition, OXA-488 and OXA-494 showed an increased affinity for penicillins, which contributed to the increased catalytic efficiency against ampicillin, especially OXA-488. Chromosomally encoded resistance mechanisms are usually overshadowed by acquired mechanisms. However, understanding their real contribution is essential to comprehend the versatile profiles verified in P. aeruginosa isolates. Such information can help to choose the best therapy in a scenario of limited options.

Proteomic Analysis Suggests Altered Mitochondrial Metabolic Profile Associated With Diabetic Cardiomyopathy.

Diabetic cardiomyopathy (DbCM) occurs independently of cardiovascular diseases or hypertension, leading to heart failure and increased risk for death in diabetic patients. To investigate the molecular mechanisms involved in DbCM, we performed a quantitative proteomic profiling analysis in the left ventricle (LV) of type 2 diabetic mice. Six-month-old C57BL/6J-lepr/lepr (db/db) mice exhibited DbCM associated with diastolic dysfunction and cardiac hypertrophy. Using quantitative shotgun proteomic analysis, we identified 53 differentially expressed proteins in the LVs of db/db mice, majorly associated with the regulation of energy metabolism. The subunits of ATP synthase that form the F1 domain, and Cytochrome c1, a catalytic core subunit of the complex III primarily responsible for electron transfer to Cytochrome c, were upregulated in diabetic LVs. Upregulation of these key proteins may represent an adaptive mechanism by diabetic heart, resulting in increased electron transfer and thereby enhancement of mitochondrial ATP production. Conversely, diabetic LVs also showed a decrease in peptide levels of NADH dehydrogenase 1ß subcomplex subunit 11, a subunit of complex I that catalyzes the transfer of electrons to ubiquinone. Moreover, the atypical kinase COQ8A, an essential lipid-soluble electron transporter involved in the biosynthesis of ubiquinone, was also downregulated in diabetic LVs. Our study indicates that despite attempts by hearts from diabetic mice to augment mitochondrial ATP energetics, decreased levels of key components of the electron transport chain may contribute to impaired mitochondrial ATP production. Preserved basal mitochondrial respiration along with the markedly reduced maximal respiratory capacity in the LVs of db/db mice corroborate the association between altered mitochondrial metabolic profile and cardiac dysfunction in DbCM.

Dexamethasone modulates immature neutrophils and interferon programming in severe COVID-19.

Although critical for host defense, innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS, compared to ARDS from other respiratory pathogens, is unclear. Moreover, mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics, we discovered that, compared to bacterial ARDS, COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils, altered IFNactive neutrophils, downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion, potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19.

The Role of Lebanon in the COVID-19 Butterfly Effect: The B.1.398 Example.

In the present study, we provide a retrospective genomic surveillance of the SARS-CoV-2 pandemic in Lebanon; we newly sequence the viral genomes of 200 nasopharyngeal samples collected between July 2020 and February 2021 from patients in different regions of Lebanon and from travelers crossing the Lebanese-Syrian border, and we also analyze the Lebanese genomic dataset available at GISAID. Our results show that SARS-CoV-2 infections in Lebanon during this period were shaped by the turnovers of four dominant SARS-CoV-2 lineages, with B.1.398 being the first to thoroughly dominate. Lebanon acted as a dispersal center of B.1.398 to other countries, with intercontinental transmissions being more common than within-continent. Within the country, the district of Tripoli, which was the source of 43% of the total B.1.398 sequences in our study, was identified as being an important source of dispersal in the country. In conclusion, our findings exemplify the butterfly effect, by which a lineage that emerges in a small area can be spread around the world, and highlight the potential role of developing countries in the emergence of new variants.