Enzymes in "Green" Synthetic Chemistry: Laccase and Lipase.

Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes and alternatives to many common catalysts. Their essential advantages are high catalytic efficiency, substrate specificity, minimal formation of byproducts, and low energy demand. All of these benefits make enzymes highly desirable targets of academic research and industrial development. This review has the modest aim of briefly overviewing the classification, mechanism of action, basic kinetics and reaction condition effects that are common across all six enzyme classes. Special attention is devoted to immobilization strategies as the main tools to improve the resistance to environmental stress factors (temperature, pH and solvents) and prolong the catalytic lifecycle of these biocatalysts. The advantages and drawbacks of methods such as macromolecular crosslinking, solid scaffold carriers, entrapment, and surface modification (covalent and physical) are discussed and illustrated using numerous examples. Among the hundreds and possibly thousands of known and recently discovered enzymes, hydrolases and oxidoreductases are distinguished by their relative availability, stability, and wide use in synthetic applications, which include pharmaceutics, food and beverage treatments, environmental clean-up, and polymerizations. Two representatives of those groups-laccase (an oxidoreductase) and lipase (a hydrolase)-are discussed at length, including their structure, catalytic mechanism, and diverse usage. Objective representation of the current status and emerging trends are provided in the main conclusions.

A large-scale genomically predicted protein mass database enables rapid and broad-spectrum identification of bacterial and archaeal isolates by mass spectrometry.

MALDI-TOF MS-based microbial identification relies on reference spectral libraries, which limits the screening of diverse isolates, including uncultured lineages. We present a new strategy for broad-spectrum identification of bacterial and archaeal isolates by MALDI-TOF MS using a large-scale database of protein masses predicted from nearly 200,000 publicly available genomes. We verify the ability of the database to identify microorganisms at the species level and below, achieving correct identification for > 90% of measured spectra. We further demonstrate its utility by identifying uncultured strains from mouse feces with metagenomics, allowing the identification of new strains by customizing the database with metagenome-assembled genomes.

Complete genome sequences of Ruminococcus torques strains JCM 36208 and JCM 36209, isolated from the feces of a healthy Japanese male.

Here, we report the complete genome sequences of two Ruminococcus torques strains (JCM 36208 and JCM 36209) that were newly isolated from the feces of a healthy Japanese male. Both genomes consist of a single circular chromosome with a length of ~2.8 Mbp and a G+C content of 41.8%.

Complete genome sequence of Butyricimonas faecihominis JCM 18676T.

We report a complete genome sequence of Butyricimonas faecihominis JCM 18676T, generated by nanopore sequencing. The genome consists of a single circular chromosome of 4,851,806 bp, with a G + C content of 42.9%, and was predicted to contain 15 rRNA and 61 tRNA genes and encode for 3,946 proteins.

Study Protocol of a Prospective, Monocentric, Single-Arm Study Investigating the Safety and Efficacy of Local Ablation of Symptomatic Uterine Fibroids with US-Guided High-Intensity Focused Ultrasound (HIFU).

Uterine fibroids are the most common benign tumors of the uterus. Approximately 20-50% of women with myomas experience a variety of symptoms such as vaginal bleeding, abdominal pain, pelvic pain and pressure, and urological problems, possibly interfering with fertility and pregnancy. Although surgery remains the standard treatment option for fibroids, non-invasive therapeutic options, such as high-intensity focused ultrasound (HIFU), have emerged over the last dec ade. During HIFU, ultrasound is focused on the target tissue causing coagulation necrosis. HIFU has, meanwhile, become an established method for treating uterine fibroids in many countries. Clinical data have shown that it effectively alleviates fibroid-related symptoms and reduces fibroid size with a very low rate of side effects. However, there is a lack of data on how this treatment affects laboratory parameters and structural features of uterine tissue. As our center is the only one in German-speaking countries where ultrasound-guided HIFU technology is currently established, the aim of this prospective, monocentric, single-arm trial is not only to evaluate the safety and efficacy of local US-guided HIFU in symptomatic uterine fibroid patients according to GCP standards but also to explore its effects on blood parameters and the structural integrity of uterine tissue using elastographic methods.

Complete genome sequence of Blautia luti JCM 17040T.

We generated a complete genome sequence of the type strain of Blautia luti (JCM 17040T = DSM 14534T) by Nanopore sequencing. The genome consists of a circular chromosome of 3,741,599 bp with a G + C content of 42.9% and was predicted to contain 3,431 protein-coding sequences.

Elucidating colorectal cancer-associated bacteria through profiling of minimally perturbed tissue-associated microbiota.

Sequencing-based interrogation of gut microbiota is a valuable approach for detecting microbes associated with colorectal cancer (CRC); however, such studies are often confounded by the effect of bowel preparation. In this study, we evaluated the viability of identifying CRC-associated mucosal bacteria through centimeter-scale profiling of the microbiota in tumors and adjacent noncancerous tissue from eleven patients who underwent colonic resection without preoperative bowel preparation. High-throughput 16S rRNA gene sequencing revealed that differences between on- and off-tumor microbiota varied considerably among patients. For some patients, phylotypes affiliated with genera previously implicated in colorectal carcinogenesis, as well as genera with less well-understood roles in CRC, were enriched in tumor tissue, whereas for other patients, on- and off-tumor microbiota were very similar. Notably, the enrichment of phylotypes in tumor-associated mucosa was highly localized and no longer apparent even a few centimeters away from the tumor. Through short-term liquid culturing and metagenomics, we further generated more than one-hundred metagenome-assembled genomes, several representing bacteria that were enriched in on-tumor samples. This is one of the first studies to analyze largely unperturbed mucosal microbiota in tissue samples from the resected colons of unprepped CRC patients. Future studies with larger cohorts are expected to clarify the causes and consequences of the observed variability in the emergence of tumor-localized microbiota among patients.

Bone Mass Changes Following Percutaneous Radiofrequency Ablation, Osteoplasty, Reinforcement, and Internal Fixation of Periacetabular Osteolytic Metastases.

BACKGROUND: The success of orthopedic interventions for periacetabular osteolytic metastases depends on the progression or regression of cancer-induced bone loss. PURPOSE: To characterize relative bone mass changes following percutaneous radiofrequency ablation, osteoplasty, cement reinforcement, and internal screw fixation (AORIF). METHODS: Of 70 patients who underwent AORIF at a single institution, 21 patients (22 periacetabular sites; average follow-up of 18.5 ± 12.3 months) had high-resolution pelvic bone CT scans, with at least one scan within 3 months following their operation (baseline) and a comparative scan at least 6 months post-operatively. In total, 73 CT scans were measured for bone mass changes using Hounsfield Units (HU). A region of interest was defined for the periacetabular area in the coronal, axial, and sagittal reformation planes for all CT scans. For 6-month and 1-year scans, the coronal and sagittal HU were combined to create a weight-bearing HU (wbHU). Three-dimensional volumetric analysis was performed on the baseline and longest available CT scans. Cohort survival was compared to predicted PathFx 3.0 survival. RESULTS: HU increased from baseline post-operative (1.2 ± 1.1 months) to most recent follow-up (20.2 ± 12.1 months) on coronal (124.0 ± 112.3), axial (140.3 ± 153.0), and sagittal (151.9 ± 162.4), p < 0.05. Grayscale volumetric measurements increased by 173.4 ± 166.4 (p < 0.05). AORIF median survival was 27.7 months (6.0 months PathFx3.0 predicted; p < 0.05). At 12 months, patients with >10% increase in wbHU demonstrated superior median survival of 36.5 months (vs. 26.4 months, p < 0.05). CONCLUSION: Percutaneous stabilization leads to improvements in bone mass and may allow for delays in extensive open reconstruction procedures.

Complete Genome Sequence of Coprobacter fastidiosus JCM 33896T.

We generated a complete genome sequence of Coprobacter fastidiosus JCM 33896T by nanopore sequencing. The genome consists of a single circular chromosome of 3,444,538 bp with a G+C content of 38.4%. Annotation predicted 15 rRNA genes, 67 tRNA genes and 2,662 protein-coding sequences.

Complete Genome Sequence of Anaerostipes hadrus JCM 17467T.

We report a complete genome sequence of Anaerostipes hadrus JCM 17467T. The genome consists of a circular chromosome of 2,804,089 bp, with a G+C content of 37.3%. The genome was predicted to contain 21 rRNA genes, 65 tRNA genes, and 2,675 protein-coding sequences.

Percutaneous Ablation, Osteoplasty, Reinforcement, and Internal Fixation for Pain and Ambulatory Function in Periacetabular Osteolytic Malignancies.

Background Osteolytic neoplasms to periacetabular bone frequently cause pain and fractures. Immediate recovery is integral to lifesaving ambulatory oncologic care and maintaining quality of life. Yet, open acetabular reconstructive surgeries are associated with numerous complications that delay cancer treatments. Purpose To determine the effectiveness for short- and long-term pain and ambulatory function following percutaneous ablation, osteoplasty, reinforcement, and internal fixation (AORIF) for periacetabular osteolytic neoplasm. Materials and Methods This retrospective observational study evaluated clinical data from 50 patients (mean age, 65 years ± 14 [SD]; 25 men, 25 women) with osteolytic periacetabular metastases or myeloma. The primary outcome of combined pain and ambulatory function index score (range, 1 [bedbound] through 10 [normal ambulation]) was assessed before and after AORIF at 2 weeks and then every 3 months up to 40 months (overall median follow-up, 11 months [IQR, 4-14 months]). Secondary outcomes included Eastern Cooperative Oncology Group (ECOG) score, infection, transfusion, 30-day readmission, mortality, and conversion hip arthroplasty. Serial radiographs and CT images were obtained to assess the hip joint integrity. The paired t test or Wilcoxon signed-rank test and Kaplan-Meier analysis were used to analyze data. Results Mean combined pain and ambulatory function index scores improved from 4.5 ± 2.4 to 7.8 ± 2.1 (P < .001) and median ECOG scores from 3 (IQR, 2-4) to 1 (IQR, 1-2) (P < .001) at the first 2 weeks after AORIF. Of 22 nonambulatory patients, 19 became ambulatory on their first post-AORIF visit. Pain and functional improvement were retained beyond 1 year, up to 40 months after AORIF in surviving patients. No hardware failures, surgical site infections, readmissions, or delays in care were identified following AORIF. Of 12 patients with protrusio acetabuli, one patient required a conversion hemiarthroplasty at 24 months. Conclusion The ablation, osteoplasty, reinforcement, and internal fixation, or AORIF, technique was effective for short- and long-term improvement of pain and ambulatory function in patients with periacetabular osteolytic neoplasm. © RSNA, 2023.

The Lack of Natural IgM Increases Susceptibility and Impairs Anti-Vi Polysaccharide IgG Responses in a Mouse Model of Typhoid.

Circulating IgM present in the body prior to any apparent Ag exposure is referred to as natural IgM. Natural IgM provides protective immunity against a variety of pathogens. Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever in humans. Because mice are not permissive to S. Typhi infection, we employed a murine model of typhoid using S. enterica serovar Typhimurium expressing the Vi polysaccharide (ViPS) of S. Typhi (S. Typhimurium strain RC60) to evaluate the role of natural IgM in pathogenesis. We found that natural mouse IgM binds to S. Typhi and S. Typhimurium. The severity of S. Typhimurium infection in mice is dependent on presence of the natural resistance-associated macrophage protein 1 (Nramp1) allele; therefore, we infected mice deficient in secreted form of IgM (sIgM) on either a Nramp1-resistant (129S) or -susceptible (C57BL/6J) background. We found that the lack of natural IgM results in a significantly increased susceptibility and an exaggerated liver pathology regardless of the route of infection or the Nramp1 allele. Reconstitution of sIgM-/- mice with normal mouse serum or purified polyclonal IgM restored the resistance to that of sIgM+/+ mice. Furthermore, immunization of sIgM-/- mice with heat-killed S. Typhi induced a significantly reduced anti-ViPS IgG and complement-dependent bactericidal activity against S. Typhi in vitro, compared with that of sIgM+/+ mice. These findings indicate that natural IgM is an important factor in reducing the typhoid severity and inducing an optimal anti-ViPS IgG response to vaccination.

Risk factors for campylobacteriosis in Australia: outcomes of a 2018-2019 case-control study.

BACKGROUND: We aimed to identify risk factors for sporadic campylobacteriosis in Australia, and to compare these for Campylobacter jejuni and Campylobacter coli infections. METHODS: In a multi-jurisdictional case-control study, we recruited culture-confirmed cases of campylobacteriosis reported to state and territory health departments from February 2018 through October 2019. We recruited controls from notified influenza cases in the previous 12 months that were frequency matched to cases by age group, sex, and location. Campylobacter isolates were confirmed to species level by public health laboratories using molecular methods. We conducted backward stepwise multivariable logistic regression to identify significant risk factors. RESULTS: We recruited 571 cases of campylobacteriosis (422 C. jejuni and 84 C. coli) and 586 controls. Important risk factors for campylobacteriosis included eating undercooked chicken (adjusted odds ratio [aOR] 70, 95% CI 13-1296) or cooked chicken (aOR 1.7, 95% CI 1.1-2.8), owning a pet dog aged < 6 months (aOR 6.4, 95% CI 3.4-12), and the regular use of proton-pump inhibitors in the 4 weeks prior to illness (aOR 2.8, 95% CI 1.9-4.3). Risk factors remained similar when analysed specifically for C. jejuni infection. Unique risks for C. coli infection included eating chicken pâté (aOR 6.1, 95% CI 1.5-25) and delicatessen meats (aOR 1.8, 95% CI 1.0-3.3). Eating any chicken carried a high population attributable fraction for campylobacteriosis of 42% (95% CI 13-68), while the attributable fraction for proton-pump inhibitors was 13% (95% CI 8.3-18) and owning a pet dog aged < 6 months was 9.6% (95% CI 6.5-13). The population attributable fractions for these variables were similar when analysed by campylobacter species. Eating delicatessen meats was attributed to 31% (95% CI 0.0-54) of cases for C. coli and eating chicken pâté was attributed to 6.0% (95% CI 0.0-11). CONCLUSIONS: The main risk factor for campylobacteriosis in Australia is consumption of chicken meat. However, contact with young pet dogs may also be an important source of infection. Proton-pump inhibitors are likely to increase vulnerability to infection.

Rare Presentation of Rosai-Dorfman Disease in Soft Tissue: Diagnostic Findings and Surgical Treatment.

Introduction and Importance. Rosai-Dorfman disease (RDD) is a rare, benign type II histiocytosis characterized by the infiltration of S100+ histiocytes and emperipolesis. The disease may present in the lymph nodes (nodal RDD), in extranodal sites, or in both nodal and extranodal sites. Among those patients who present exclusively in extranodal sites, only a minority of cases present in the soft tissue. Case Presentation. An 18-year-old female presented to orthopedic oncology clinic with a chief complaint of a mass located in her lower back. The patient underwent excision of the lumbosacral mass. Pathologic review demonstrated emperipolesis of lymphocytes and plasma cells within enlarged, eosinophilic histiocytes in a background of lymphoplasmacytic infiltration and collagenous stroma. Immunohistochemical staining demonstrated S100+ and CD163+ histiocytes, consistent with diagnosis of soft tissue RDD. Clinical Discussion. Histologically, RDD is generally characterized by emperipolesis-the presence of intact lymphocytes within the histiocyte cytoplasm-and a mixed infiltrate of S100+ histiocytes, mononuclear cells, plasma cells, and lymphocytes. Although soft tissue RDD may histologically resemble nodal RDD, soft tissue RDD also demonstrates some notable histologic differences including the lack of nodal architecture, the presence of increased fibrosis and collagen deposition, and generally fewer RDD cells. Conclusion. This case presentation demonstrates one few reports of isolated soft tissue RDD within the lumbosacral region without associated lymphadenopathy or skin changes and highlights the heterogeneity that still exists in the treatment paradigm of extranodal RDD.

Characterization and Demonstration of Mock Communities as Control Reagents for Accurate Human Microbiome Community Measurements.

Standardization and quality assurance of microbiome community analysis by high-throughput DNA sequencing require widely accessible and well-characterized reference materials. Here, we report on newly developed DNA and whole-cell mock communities to serve as control reagents for human gut microbiota measurements by shotgun metagenomics and 16S rRNA gene amplicon sequencing. The mock communities were formulated as near-even blends of up to 20 bacterial species prevalent in the human gut, span a wide range of genomic guanine-cytosine (GC) contents, and include multiple strains with Gram-positive type cell walls. Through a collaborative study, we carefully characterized the mock communities by shotgun metagenomics, using previously developed standardized protocols for DNA extraction and sequencing library construction. Further, we validated fitness of the mock communities for revealing technically meaningful differences among protocols for DNA extraction and metagenome/16S rRNA gene amplicon library construction. Finally, we used the mock communities to reveal varying performance of metagenome-based taxonomic profilers and the impact of trimming and filtering of sequencing reads on observed species profiles. The latter showed that aggressive preprocessing of reads may result in substantial GC-dependent bias and should thus be carefully evaluated to minimize unintended effects on species abundances. Taken together, the mock communities are expected to support a myriad of applications that rely on well-characterized control reagents, ranging from evaluation and optimization of methods to assessment of reproducibility in interlaboratory studies and routine quality control. IMPORTANCE Application of high-throughput DNA sequencing has greatly accelerated human microbiome research and its translation into new therapeutic and diagnostic capabilities. Microbiome community analyses results can, however, vary considerably across studies or laboratories, and establishment of measurement standards to improve accuracy and reproducibility has become a priority. The here-developed mock communities, which are available from the NITE Biological Resource Center (NBRC) at the National Institute of Technology and Evaluation (NITE, Japan), provide well-characterized control reagents that allow users to judge the accuracy of their measurement results. Widespread and consistent adoption of the mock communities will improve reproducibility and comparability of microbiome community analyses, thereby supporting and accelerating human microbiome research and development.

Enzymatic Synthesis and Antimicrobial Activity of Oligomer Analogues of Medicinal Biopolymers from Comfrey and Other Species of the Boraginaceae Family.

This study reports the first enzymatic synthesis leading to several oligomer analogues of poly[3-(3,4-dihydroxyphenyl)glyceric acid]. This biopolymer, extracted from plants of the Boraginaceae family has shown a wide spectrum of pharmacological properties, including antimicrobial activity. Enzymatic ring opening polymerization of 2-methoxycarbonyl-3-(3,4-dibenzyloxyphenyl)oxirane (MDBPO) using lipase from Candida rugosa leads to formation of poly[2-methoxycarbonyl-3-(3,4-dibenzyloxyphenyl)oxirane] (PMDBPO), with a degree of polymerization up to 5. Catalytic debenzylation of PMDBPO using H2 on Pd/C yields poly[2-methoxycarbonyl-3-(3,4-dihydroxyphenyl)oxirane] (PMDHPO) without loss in molecular mass. Antibacterial assessment of natural polyethers from different species of Boraginaceae family Symhytum asperum, S. caucasicum,S. grandiflorum, Anchusa italica, Cynoglossum officinale, and synthetic polymers, poly[2-methoxycarbonyl-3-(3,4-dimethoxyphenyl)oxirane (PMDMPO) and PMDHPO, reveals that only the synthetic analogue produced in this study (PMDHPO) exhibits a promising antimicrobial activity against pathogenic strains S.aureus ATCC 25923 and E.coli ATCC 25922 the minimum inhibitory concentration (MIC) being 100 µg/mL.

A Prospective, Open-Label Pilot Study of Concurrent Male Partner Treatment for Bacterial Vaginosis.

Up to 50% of women receiving first-line antibiotics for bacterial vaginosis (BV) experience recurrence within 12 weeks. Evidence suggests that reinfection from an untreated regular sexual partner contributes to recurrence. We conducted a pilot study of 34 heterosexual couples to describe the impact of concurrent partner treatment on the composition of the genital microbiota over a 12-week period. We also determined the acceptability and tolerability of concurrent partner treatment and obtained preliminary estimates of the efficacy of the intervention to inform a randomized controlled trial (RCT). Women received first-line antibiotic treatment for BV (i.e., oral metronidazole or intravaginal clindamycin), and their male partner received oral metronidazole, 400 mg, and 2% clindamycin cream applied topically to penile skin, both twice daily for 7 days. The genital microbiota was characterized at three anatomical sites (women, vaginal; men, cutaneous penile and first-pass urine [representing the urethra]) using 16S rRNA gene sequencing. Immediately posttreatment, concurrent partner treatment significantly reduced the abundance of BV-associated bacteria (false-discovery rate [FDR] corrected P value < 0.05) and altered the overall microbiota composition of all three anatomical sites (P = 0.001). Suppression of BV-associated bacteria was sustained in the majority (81%) of women over the 12-week period (FDR P value < 0.05), despite BV-associated bacteria reemerging at both genital sites in men. In this cohort of women at high risk for recurrence, five recurred within 12 weeks of treatment (17%; 95% confidence interval [CI], 6 to 34%). Importantly, men tolerated and adhered to combination therapy. Our findings provide support for an RCT of combined oral and topical male partner treatment for BV. IMPORTANCE Recurrence of BV following standard treatment is unacceptably high. Posttreatment recurrence is distressing for women, and it imposes a considerable burden on the health care system. Recurrences result in multiple presentations to clinical services and repeated antibiotic use, and the associated obstetric and gynecological sequelae are significant. New treatments to improve long-term BV cure are urgently needed. Here, we used 16S rRNA gene sequencing to investigate changes in the microbiota composition at three genital sites (vagina, penile skin, and male urethra) of heterosexual couples undergoing concurrent partner treatment for bacterial vaginosis (BV). We found that concurrent partner treatment immediately and significantly altered the composition of the genital microbiota of both partners, with a reduction in BV-associated bacteria seen at all three sites. BV cure at 12 weeks posttreatment was higher than expected. These microbiological data provide evidence for continued investigation of partner treatment as a strategy to improve BV cure.

Multi-Omics Analysis to Generate Hypotheses for Mild Health Problems in Monkeys.

Certain symptoms associated with mild sickness and lethargy have not been categorized as definitive diseases. Confirming such symptoms in captive monkeys (Macaca fascicularis, known as cynomolgus monkeys) can be difficult; however, it is possible to observe and analyze their feces. In this study, we investigated the relationship between stool state and various omics data by considering objective and quantitative values of stool water content as a phenotype for analysis. By examining the food intake of the monkeys and assessing their stool, urine, and plasma, we attempted to obtain a comprehensive understanding of the health status of individual monkeys and correlate it with the stool condition. Our metabolomics data strongly suggested that many lipid-related metabolites were correlated with the stool water content. The lipidomic analysis revealed the involvement of saturated and oxidized fatty acids, metallomics revealed the contribution of selenium (a bio-essential trace element), and intestinal microbiota analysis revealed the association of several bacterial species with the stool water content. Based on our results, we hypothesize that the redox imbalance causes minor health problems. However, it is not possible to make a definite conclusion using multi-omics alone, and other hypotheses could be proposed.

Increased Production of Outer Membrane Vesicles by Salmonella Interferes with Complement-Mediated Innate Immune Attack.

Bacterial outer membrane vesicles (OMVs) enriched with bioactive proteins, toxins, and virulence factors play a critical role in host-pathogen and microbial interactions. The two-component system PhoP-PhoQ (PhoPQ) of Salmonella enterica orchestrates the remodeling of outer membrane lipopolysaccharide (LPS) molecules and concomitantly upregulates OMV production. In this study, we document a novel use of nanoparticle tracking analysis to determine bacterial OMV size and number. Among the PhoPQ-activated genes tested, pagC expression had the most significant effect on the upregulation of OMV production. We provide the first evidence that PhoPQ-mediated upregulation of OMV production contributes to bacterial survival by interfering with complement activation. OMVs protected bacteria in a dose-dependent manner, and bacteria were highly susceptible to complement-mediated killing in their absence. OMVs from bacteria expressing PagC bound to complement component C3b in a dose-dependent manner and inactivated it by recruiting complement inhibitor Factor H. As we also found that Factor H binds to PagC, we propose that PagC interferes with complement-mediated killing of Salmonella in the following two steps: first by engaging Factor H, and second, through the production of PagC-enriched OMVs that divert and inactivate the complement away from the bacteria. Since PhoPQ activation occurs intracellularly, the resultant increase in PagC expression and OMV production is suggested to contribute to the local and systemic spread of Salmonella released from dying host cells that supports the infection of new cells. IMPORTANCE Bacterial outer membrane vesicles (OMVs) mediate critical bacterium-bacterium and host-microbial interactions that influence pathogenesis through multiple mechanisms, including the elicitation of inflammatory responses, delivery of virulence factors, and enhancement of biofilm formation. As such, there is a growing interest in understanding the underlying mechanisms of OMV production. Recent studies have revealed that OMV biogenesis is a finely tuned physiological process that requires structural organization and selective sorting of outer membrane components into the vesicles. In Salmonella, outer membrane remodeling and OMV production are tightly regulated by its PhoPQ system. In this study, we demonstrate that PhoPQ-regulated OMV production plays a significant role in defense against host innate immune attack. PhoPQ-activated PagC expression recruits the complement inhibitor Factor H and degrades the active C3 component of complement. Our results provide valuable insight into the combination of tools and environmental signals that Salmonella employs to evade complement-mediated lysis, thereby suggesting a strong evolutionary adaptation of this facultative intracellular pathogen to protect itself during its extracellular stage in the host.

Microdroplet-based system for culturing of environmental microorganisms using FNAP-sort.

Droplet microfluidics has emerged as a powerful technology for improving the culturing efficiency of environmental microorganisms. However, its widespread adoption has been limited due to considerable technical challenges, especially related to identification and manipulation of individual growth-positive droplets. Here, we combined microfluidic droplet technology with on-chip "fluorescent nucleic acid probe in droplets for bacterial sorting" (FNAP-sort) for recovery of growth-positive droplets and droplet microdispensing to establish an end-to-end workflow for isolation and culturing of environmental microbes. As a proof-of-concept, we demonstrate the ability of our technique to yield high-purity cultures of rare microorganisms from a representative complex environmental microbiome. As our system employs off-the-shelf commercially available equipment, we believe that it can be readily adopted by others and may thus find widespread use toward culturing the high proportion of as-of-yet uncultured microorganisms in different biomes.