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Effective bioremediation of hydrocarbons requires innovative approaches to minimize phosphate precipitation in soils of different buffering capacities. Understanding the mechanisms underlying sustained stimulation of bacterial activity remains a key challenge for optimizing bioremediation—particularly in northern regions. Positron emission tomography (PET) can trace microbial activity within the naturally occurring soil structure of intact soils. Here, we use PET to test two hypotheses: (1) optimizing phosphate bioavailability in soil will outperform a generic biostimulatory solution in promoting hydrocarbon remediation and (2) oligotrophic biostimulation will be more effective than eutrophic approaches. In so doing, we highlight the key bacterial taxa that underlie aerobic and anaerobic hydrocarbon degradation in subarctic soils. In particular, we showed that (i) optimized phosphate bioavailability outperformed generic biostimulatory solutions in promoting hydrocarbon degradation, (ii) oligotrophic biostimulation is more effective than eutrophic approaches, and (iii) optimized biostimulatory solutions stimulated specific soil regions and bacterial consortia. The knowledge gleaned from this study will be crucial in developing field-scale biodegradation treatments for sustained stimulation of bacterial activity in northern regions.

Blinded, site-independent (remote) ratings from audio-digital recordings of site-based Positive and Negative Syndrome Scale (PANSS) interviews were obtained in a 5-week, randomized, double-blinded study assessing the safety, tolerability, and efficacy of KarXT (a fixed combination of xanomeline and trospium chloride) in hospitalized adults with schizophrenia experiencing an acute exacerbation of psychosis (EMERGENT-1; ClinicalTrials.gov identifier: NCT3697252). The blinded site-independent raters had no knowledge of site location, study visit, drug vs. placebo assignment, or any treatment emergent adverse events (TEAEs). Concordance analyses of 561 paired site-based and site-independent PANSS ratings across all visits revealed a high correlation (ICC = 0.775). Paired scoring differences were positively correlated with the PANSS total score (Spearman's rho = 0.37, p 0.0001). Paired PANSS scores were available from 148 subjects at both the baseline and end of study visits (KarXT = 72, Placebo = 76). Site-based PANSS total scores (primary aim) revealed a significantly greater improvement from baseline in the KarXT group compared to the placebo group (p 0.0001). The blinded site-independent PANSS total scores derived from listening to and scoring the recorded site-based PANSS interviews replicated this finding (p 0.001) and yielded an overall predictive value of 85.1% for matching the site-based response/non-response outcomes. TEAE's have the potential to "unblind" site-based ratings. In this study, the site-independent raters were blinded to TEAEs, affirmed the site-based PANSS ratings, and mitigated concerns about possible functional unblinding of site-based raters. This method of blinded assessment via audio-digital recordings may have utility for other studies concerned with ratings precision and/or functional unblinding.

Aim Redo ileocolic resection in patients with Crohn's disease (CD) is associated with significant technical challenges that may be associated with high complication rates. The aim of this study was to evaluate the feasibility of near-infrared fluorescence angiography with indocyanine green (ICG), often used to evaluate blood supply to the anastomosis in CD patients undergoing repeat ileocolic resection. Method This study was a retrospective analysis of patients who underwent redo ileocolic resection using ICG bowel perfusion assessment between 2015 and 2021. Patients were matched and compared on a 1:2 basis with a control group undergoing the same procedure without perfusion assessment. Results Twelve patients underwent redo ileocolic resection with ICG perfusion assessment (ICG group). These were compared with 24 patients who underwent the procedure without ICG (control group). Both groups were similar in demographics and operative characteristics including median operating time (255 vs. 255.5 min, p = 0.39) and conversion rate (22% vs. 36.8%, p = 0.68). Median estimated blood loss was significantly higher in the ICG group [150 (50-400) vs. 100 ml (20-125)]. Successful ICG perfusion assessment was seen in all patients in the ICG group and did not change management in any case. Overall postoperative complication rates were comparable between the groups (58.3% vs. 54.1%, p = 0.72). No anastomotic leaks occurred in the ICG group compared with one (1/24, 4.2%) in the control group (p = 0.99). Conclusion Fluorescence ICG perfusion assessment is feasible and safe in redo ileocolic resection in patients with CD. Larger studies are needed to evaluate whether this technique should be routinely used in these complex surgical interventions.

Purpose To define injection index (II) and assess its impact on visual acuity (VA) in pigment epithelial detachment from age-related macular degeneration over 5 years. Methods Injection index is defined as the mean anti-vascular endothelial growth factor injections per year from presentation. A retrospective study of 256 eyes in 213 patients was performed. Patients were stratified by II (high: ≥9, low: Results Baseline characteristics showed no differences across II groups. Mean (range) follow-up, in years, was 5.02 (1.04-12.74) for all patients. Mean logMAR VA (Snellen VA) were 0.60 (20/80) and 0.56 (20/73) at baseline, 0.52 (20/66) and 0.59 (20/78) at Year 1, 0.45 (20/56) and 0.67 (20/94) at Year 2, 0.38 (20/48) and 0.66 (20/91) at Year 3, 0.41 (20/51) and 0.89 (20/155) at Year 4, and 0.35 (20/45) and 0.79 (20/123) at Year 5 for the high and low II groups, respectively. Linear regression analysis showed a gain of 0.5 approxETDRS letters with each additional injection per year. Conclusion Increased II was associated with better mean VA, suggesting that long-term continuous vascular endothelial growth factor suppression may improve VA in eyes thought to carry poor prognoses.

Microviridins, tricyclic peptide natural products originally isolated from cyanobacteria, function as inhibitors of diverse serine-type proteases. Here we report the structure and biochemical characterization of AMdnB, a unique iterative macrocyclase involved in a microviridin biosynthetic pathway from Anabaena sp. PCC 7120. The ATP-dependent cyclase, along with the homologous AMdnC, introduce up to nine macrocyclizations on three distinct core regions of a precursor peptide, AMdnA. The results presented here provide structural and mechanistic insight into the iterative chemistry of AMdnB. In vitro AMdnB-catalyzed cyclization reactions demonstrate the synthesis of the two predicted tricyclic products from a multi-core precursor peptide substrate, consistent with a distributive mode of catalysis. The X-ray structure of AMdnB shows a structural motif common to ATP-grasp cyclases involved in RiPPs biosynthesis. Additionally, comparison with the non-iterative MdnB allows insight into the structural basis for the iterative chemistry. Overall, the presented results provide insight into the general mechanism of iterative enzymes in ribosomally synthesized and post-translationally modified peptide biosynthetic pathways.

Microbial degradation of subsurface organic contaminants is often hindered by the low availability of both contaminants and nutrients, especially phosphorus (P). The use of activated carbon and traditional P fertilizers to overcome these challenges has proved ineffective; therefore, we sought to find an innovative and effective solution. By heating bone meal-derived organic residues in water in a closed reactor, we synthesized nonporous colloids composed of aromatic and aliphatic structures linked to P groups. X-ray absorption near edge spectroscopy analysis revealed that the materials contain mostly bioavailable forms of P (i.e., adsorbed P and magnesium-bearing brushite). The capacity of the materials to adsorb organic contaminants was investigated using benzene and batch isotherm experiments. The adsorption isotherms were fitted to the linearized Freundlich model; isotherm capacity (logKF ) values for the materials ranged between 1.6 and 2.8 μg g-1 . These results indicate that the colloidal materials have a high affinity for organic contaminants. This, coupled with their possession of bioavailable P, should make them effective amendments for in situ groundwater bioremediation. Also, the materials' chemical properties suggest that they are not recalcitrant, implying that they will not become potential contaminants when released into the environment.

Queuosine is a structurally unique and functionally important tRNA modification, widely distributed in eukaryotes and bacteria. The final step of queuosine biosynthesis is the reduction/deoxygenation of epoxyqueuosine to form the cyclopentene motif of the nucleobase. The chemistry is performed by the structurally and functionally characterized cobalamin-dependent QueG. However, the queG gene is absent from several bacteria that otherwise retain queuosine biosynthesis machinery. Members of the IPR003828 family (previously known as DUF208) have been recently identified as nonorthologous replacements of QueG, and this family was renamed QueH. Here, we present the structural characterization of QueH from Thermotoga maritima. The structure reveals an unusual active site architecture with a [4Fe-4S] metallocluster along with an adjacent coordinated iron metal. The juxtaposition of the cofactor and coordinated metal ion predicts a unique mechanism for a two-electron reduction/deoxygenation of epoxyqueuosine. To support the structural characterization, in vitro biochemical and genomic analyses are presented. Overall, this work reveals new diversity in the chemistry of iron/sulfur-dependent enzymes and novel insight into the last step of this widely conserved tRNA modification.

DISCLOSURES: Funding for this summary was contributed by Arnold Ventures, California Health Care Foundation, The Donaghue Foundation, Harvard Pilgrim Health Care, and Kaiser Foundation Health Plan ...

Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700.

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multipleturnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been characterized – but only from archaeal methanogens that are anaerobic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

Zwitterionic carbohydrate modifications, such as phosphoethanolamine (PEtN), govern host-pathogen interactions. Whereas it is recognized that these modifications stimulate the host immune system, the purpose of PEtN modification remains largely descriptive. As an enabling step toward studying this carbohydrate modification, we report a synthesis of the P. temperata zwitterionic trisaccharide repeating unit. The 32-step synthesis was enabled by H-phosphonate chemistry to install the PEtN arm on a poorly reactive and sterically hindered C4-alcohol.

Few classes of natural products rival the structural audacity of oligosaccharides. Their complexity, however, has stood as an immense roadblock to translational research, as access to homogeneous material from nature is challenging. Thus, while carbohydrates are critical to the myriad functional and structural aspects of the biological sciences, their behavior is largely descriptive. This challenge presents an attractive opportunity for synthetic chemistry, particularly in the area of human milk science. First, there is an inordinate need for synthesizing homogeneous human milk oligosaccharides (HMOs). Superimposed on this goal is the mission of conducting syntheses at scale to enable animal studies. Herein, we present a personalized rumination of our involvement, and that of our colleagues, which has led to the synthesis and characterization of HMOs and mechanistic probes. Along the way, we highlight chemical, chemoenzymatic, and synthetic biology based approaches. We close with a discussion on emergent challenges and opportunities for synthesis, broadly defined, in human milk science.

Microviridins are cyanobacterial tricyclic depsipeptides with unique ring architectures and function as serine protease inhibitors. In this study, we explore two strategies to probe the structure and mechanism of macrocyclases involved in microviridin biosynthesis. The results both provide approaches for in vitro chemoenzymatic synthesis and insight into the molecular interactions and function of the biosynthetic enzymes. The first strategy involves generating constitutively activated macrocyclases whereby the leader portion of the substrate peptide is covalently attached to the ATP-grasp ligases to examine leader peptide/enzyme interactions. The second strategy uses a structure-based design to create disulfide cross-linked peptide/enzyme complexes. Together, the strategies provide constitutively active enzymes and tools to study the catalysis of the macrocyclizations on synthetic core peptides.

Elucidating the multifaceted processes of molecular activation and subsequent reactions gives a fundamental view into the development of iridium catalysts as they apply to fuels and propellants, for example, for spacecraft thrusters. Hydroxylamine, a component of the well-known hydroxylammonium nitrate (HAN) ionic liquid, is a safer alternative and mimics the chemistry and performance standards of hydrazine. The activation of hydroxylamine by anionic iridium clusters, Irn- (n = 1-5), depicts a part of the mechanism, where two hydrogen atoms are removed, likely as H2, and Irn(NOH)- clusters remain. The significant photoelectron spectral differences between these products and the bare clusters illustrate the substantial electronic changes imposed by the hydroxylamine fragment on the iridium clusters. In combination with DFT calculations, a preliminary reaction mechanism is proposed, identifying the possible intermediate steps leading to the formation of Ir(NOH)-.

The synthesis of glycosides incorporating basic nitrogen atoms is a longstanding challenge in organic synthesis. By leveraging the reactivity of glycosyl anions, a general method has been developed that enables use of glycosyl donors incorporating unprotected, basic amines.

The ability of the vagina to contract gives rise to a set of active mechanical properties that contribute to the complex function of this organ in-vivo. Regional differences in the morphology of the vagina have been long recognized, but the large heterogeneous deformations that the vagina experiences during contractions have never been quantified. Furthermore, there is no consensus regarding differences in contractility along the two primary anatomical directions of the vagina: the longitudinal direction (LD) and the circumferential direction (CD). In this study, square vaginal specimens from healthy virgin rats ( n = 15 ) were subjected to isometric planar biaxial tests at four equi-biaxial stretches of 1.0, 1.1, 1.2, and 1.3. Contractions were induced at each stretch by a high concentration potassium solution. The digital image correlation method was used to perform full-field strain measurements during contractions. The vagina was found to undergo significantly higher compressive strains, tensile strains, and contractile forces along the LD than along the CD during contractions. Specifically, when computed over all the applied equi-biaxial stretches, mean ( ± std. dev.) absolute maximum compressive strains were -(13.43 ± 1.56) % along the LD and -(3.19 ± 0.25) % along the CD, mean absolute maximum tensile strains were (10.92 ± 1.73) % along the LD and (3.62 ± 0.57) % along the CD, and mean maximum contractile forces were 6.24 ± 0.55 mN along the LD and 3.35 ± 0.56 mN along the CD. Moreover, the vaginal tissue appeared to undergo compression in the proximal region and tension in the distal region while kept at constant equi-biaxial stretches. The active mechanical properties of the healthy vagina need to be fully investigated so that detrimental alterations in vaginal contractility, such as those caused by pelvic floor disorders and current treatment strategies, can be prevented. Statement of significance Contractile forces of the vagina have been measured by several investigators using uniaxial tensile testing methods. Unlike previous studies, in this study planar-biaxial tests of vaginal specimens were performed while the full-field strains of the vagina, as induced by smooth muscle contraction, were measured. The vagina was found to generate significantly larger contractile strains and forces in the longitudinal direction than in the circumferential direction. Knowledge of the contractile mechanics of the healthy vagina is essential to understand the detrimental effects that pelvic organ prolapse and the use of surgical meshes have on the functionality of smooth muscle in the vagina.

The Fanconi anemia (FA) pathway safeguards genomic stability through cell cycle regulation and DNA damage repair. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established, but their function in mitosis is incompletely understood. Here, we performed a kinome‐wide synthetic lethality screen in FANCA−/− fibroblasts, which revealed multiple mitotic kinases as necessary for survival of FANCA‐deficient cells. Among these kinases, we identified the depletion of the centrosome kinase SIK2 as synthetic lethal upon loss of FANCA. We found that FANCA colocalizes with SIK2 at multiple mitotic structures and regulates the activity of SIK2 at centrosomes. Furthermore, we found that loss of FANCA exacerbates cell cycle defects induced by pharmacological inhibition of SIK2, including impaired G2‐M transition, delayed mitotic progression, and cytokinesis failure. In addition, we showed that inhibition of SIK2 abrogates nocodazole‐induced prometaphase arrest, suggesting a novel role for SIK2 in the spindle assembly checkpoint. Together, these findings demonstrate that FANCA‐deficient cells are dependent upon SIK2 for survival, supporting a preclinical rationale for targeting of SIK2 in FA‐disrupted cancers.

Human cardiac β myosin undergoes the crossbridge cycle as part of the force-generating mechanism of cardiac muscle. The recovery stroke is considered one of the key steps of the kinetic cycle as it is the conformational rearrangement required to position the active site residues for hydrolysis of ATP and interaction with actin. We explored the free-energy surface of the transition and investigated the effect of the genetic cardiomyopathy causing mutations R453C, I457T, and I467T on this step using metadynamics. This work extends previous studies on Dictyostelium myosin II with engineered mutations. Here, like previously, we generated an unbiased thermodynamic ensemble of reactive trajectories for the chemical step using transition path sampling. Our methodologies were able to predict the changes to the dynamics of the recovery stroke as well as predict the pathway of breakdown of ATP to ADP and HPO(4)(2−) with the stabilization of the metaphosphate intermediate. We also observed clear differences between the Dictyostelium myosin II and human cardiac β myosin for ATP hydrolysis as well as predict the effect of the mutation I467T on the chemical step.

Constitutive MAPK activation silences genes required for iodide uptake and thyroid hormone biosynthesis in thyroid follicular cells. Accordingly, most BRAFV600E papillary thyroid cancers (PTC) are refractory to radioiodide (RAI) therapy. MAPK pathway inhibitors rescue thyroid-differentiated properties and RAI responsiveness in mice and patient subsets with BRAFV600E-mutant PTC. TGFB1 also impairs thyroid differentiation and has been proposed to mediate the effects of mutant BRAF. We generated a mouse model of BRAFV600E-PTC with thyroid-specific knockout of the Tgfbr1 gene to investigate the role of TGFB1 on thyroid-differentiated gene expression and RAI uptake in vivo. Despite appropriate loss of Tgfbr1, pSMAD levels remained high, indicating that ligands other than TGFB1 were engaging in this pathway. The activin ligand subunits Inhba and Inhbb were found to be overexpressed in BRAFV600E-mutant thyroid cancers. Treatment with follistatin, a potent inhibitor of activin, or vactosertib, which inhibits both TGFBR1 and the activin type I receptor ALK4, induced a profound inhibition of pSMAD in BRAFV600E-PTCs. Blocking SMAD signaling alone was insufficient to enhance iodide uptake in the setting of constitutive MAPK activation. However, combination treatment with either follistatin or vactosertib and the MEK inhibitor CKI increased 124I uptake compared to CKI alone. In summary, activin family ligands converge to induce pSMAD in Braf-mutant PTCs. Dedifferentiation of BRAFV600E-PTCs cannot be ascribed primarily to activation of SMAD. However, targeting TGFβ/activin-induced pSMAD augmented MAPK inhibitor effects on iodine incorporation into BRAF tumor cells, indicating that these two pathways exert interdependent effects on the differentiation state of thyroid cancer cells.

Leflunomide (Lef) is an agent used in autoimmune disorders that interferes with DNA synthesis. De Novo pyrimidine synthesis is a mechanism of Gemcitabine (Gem) resistance in pancreatic cancer. This study aims to assess the efficacy and changes in the tumor microenvironment of Lef monotherapy and in combination with Gem, in a syngeneic mouse model of pancreatic cancer. Methods: MTS proliferation assays were conducted to assess growth inhibition by Gem (0-20 nM), Lef (0-40 uM) and Gem+Lef in KPC (KrasLSL.G12D/+;p53R172H/+; PdxCretg/+) cells in vitro. An in vivo heterotopic KPC model was used and cohorts were treated with: PBS (control), Gem (75 mg/kg/q3d), Lef (40 mg/kg/d), or Gem+Lef. At d28 post-treatment, tumor burden, proliferation index (Ki67), and vascularity (CD31) were measured. Changes in the frequency of peripheral and intratumoral immune cell subsets were evaluated via FACS. Liquid chromatography-mass spectrometry was used for metabolomics profiling. Results: Lef inhibits KPC cell growth and synergizes with Gem in vitro (P

BACKGROUND AND AIMS Existing therapeutic approaches to treat cholangiocarcinoma (CCA) have limited effectiveness, prompting further study to develop therapies for CCA. We report a mechanistic role for the heparan sulfate editing enzyme sulfatase 2 (SULF2) in CCA pathogenesis. APPROACH AND RESULTS In silico analysis revealed elevated SULF2 expression in human CCA samples, occurring partly through gain of SULF2 copy number. We examined the effects of knockdown or overexpression of SULF2 on tumor growth, chemoresistance, and signaling pathway activity in human CCA cell lines in vitro. Up-regulation of SULF2 in CCA leads to increased platelet-derived growth factor receptor beta (PDGFRβ)-Yes-associated protein (YAP) signaling activity, promoting tumor growth and chemotherapy resistance. To explore the utility of targeting SULF2 in the tumor microenvironment for CCA treatment, we tested an anti-SULF2 mouse monoclonal antibody, 5D5, in a mouse CCA xenograft model. Targeting SULF2 by monoclonal antibody 5D5 inhibited PDGFRβ-YAP signaling and tumor growth in the mouse xenograft model. CONCLUSIONS These results suggest that SULF2 monoclonal antibody 5D5 or related agents may be potentially promising therapeutic agents in CCA.

The movement of tropomyosin over filamentous actin regulates the cross-bridge cycle of the thick with thin filament of cardiac muscle by blocking and revealing myosin binding sites. Tropomyosin exists in three, distinct equilibrium states with one state blocking myosin-actin interactions (blocked position) and the remaining two allowing for weak (closed position) and strong myosin binding (open position). However, experimental information illuminating how myosin binds to the thin filament and influences tropomyosin’s transition across the actin surface is lacking. Using metadynamics, we mimic the effect of a single myosin head binding by determining the work required to pull small segments of tropomyosin toward the open position in several distinct regions of the thin filament. We find differences in required work due to the influence of cardiac troponin T lead to preferential binding sites and determine the mechanism of further myosin head recruitment.

Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the “CTD code” that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS)26 of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4–5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity., Namitz, Zheng et al. identify a bivalent interaction by the yeast Ess1 with CTD peptides of RNA polymerase II. Their results suggest an anchored mechanism of isomerization, and raise the possibility of eukaryotic parvulin-class prolyl isomerases gaining a broader substrate specificity during evolution, by acquiring a flexible linker that generates a more dynamic binding mode.

VicRK (WalRK or YycFG) is a conserved 2-component regulatory system (TCS) that regulates cell division, cell wall biosynthesis, and homeostasis in low-GC Gram-positive bacteria. VicRK is also associated with biofilm formation of Streptococcus mutans on the tooth surface as it directly regulates the extracellular polysaccharide (EPS) synthesis. Of the 2 components, VicK possesses both autokinase and phosphatase activities, which regulate the phosphorylation and dephosphorylation of the regulator VicR in response to environmental cues. However, the dual mechanism of VicK as the autokinase/phosphatase in regulating S. mutans’ responses is not well elucidated. Previously, it has been shown that the phosphatase activity depends on the PAS domain and residues in the DHp domain of VicK in S. mutans. Specifically, mutating proline at 222 in the PAS domain inhibits VicK phosphatase activity. We generated a VicKP222A mutant to determine the level of VicR-P in the cytoplasm by Phos-tag sodium dodecyl sulfate polyacrylamide gel electrophoresis. We show that in VicKP222A phosphatase, attenuation increased phosphorylated VicR (VicR-P) that downregulated glucosyltransferases, gtfBC, thereby reducing the synthesis of water-insoluble polysaccharides (WIS-EPS) in the biofilm. In addition, VicKP222A presented as long-rod cells, reduced growth, and displayed asymmetrical division. A major adhesin of S. mutans, SpaP was downregulated in VicKP222A, making it unable to agglutinate in saliva. In summary, we have confirmed that VicK phosphatase activity is critical to maintain optimal phosphorylation status of VicR in S. mutans, which is important for cell growth, cell division, EPS synthesis, and bacterial agglutination in saliva. Hence, VicK phosphatase activity may represent a promising target to modulate S. mutans’ pathogenicity.

Necrotizing enterocolitis (NEC) is a manifestation of maladaptive intestinal responses in preterm infants centrally medicated by unattenuated inflammation. Early in the postnatal period, preterm infants develop a deficit in arachidonic and docosahexaenoic acid, both potent regulators of inflammation. We hypothesized that the fatty acid composition of parenteral lipid emulsions uniquely induces blood and intestinal fatty acid profiles which, in turn, modifies the risk of NEC development. Forty-two preterm pigs were randomized to receive one of three lipid emulsions containing 100% soybean oil (SO), 15% fish oil (MO15), or 100% fish oil (FO100) with enteral feedings over an 8-day protocol. Blood and distal ileum tissue were collected for fatty acid analysis. The distal ileum underwent histologic, proteomic, and metabolomic analyses. Eight pigs [3/14 SO (21%), 3/14 MO15 (21%), and 2/14 FO100 (14%)] developed NEC. No differences in NEC risk were evident between groups despite differences in induced fatty acid profiles in blood and ileal tissue. Metabolomic analysis of NEC versus no NEC tissue revealed differences in tryptophan metabolism and arachidonic acid-containing glycerophospholipids. Proteomic analysis demonstrated no differences by lipid group; however, 15 proteins differentiated NEC versus no NEC in the domains of tissue injury, glucose uptake, and chemokine signaling. Exposure to parenteral lipid emulsions induces unique intestinal fatty acid and metabolomic profiles; however, these profiles are not linked to a difference in NEC development. Metabolomic and proteomic analyses of NEC versus no NEC intestinal tissue provide mechanistic insights into the pathogenesis of NEC in preterm infants. NEW & NOTEWORTHY Exposure to parenteral lipid emulsions induces unique intestinal fatty acid and metabolomic profiles; however, these profiles are not linked to a difference in NEC risk in preterm pigs. Metabolomic and proteomic analyses provide mechanistic insights into NEC pathogenesis. Compared with healthy ileal tissue, metabolites in tryptophan metabolism and arachidonic acid-containing glycerophospholipids are increased in NEC tissue. Proteomic analysis differentiates NEC versus no NEC in the domains of tissue injury, glucose uptake, and chemokine signaling.