Unswitched memory B cell deficiency in children with sickle cell disease and response to pneumococcal polysaccharide vaccine.

Early mortality in sickle cell disease (SCD) is attributed to increased infections due to loss of splenic function. Marginal zone B cells are important for initial opsonization of pathogens and can be absent in spleen histopathology in SCD. The frequency of unswitched memory B cells (UMBC), the circulating correlate of marginal zone B cells, reflects the immunologic function of the spleen. We hypothesized that asplenia in SCD is associated with alterations in the peripheral blood lymphocyte population and explored whether UMBC deficiency was associated with a clinical phenotype. We analyzed B cell subsets and clinical history for 238 children with SCD and 63 controls. The median proportion of UMBCs was lower in children with SCD compared with controls (4.7% vs. 6.6%, p < .001). Naïve B cells were higher in SCD compared with controls (80.6 vs. 76.3%, respectively, p = .02). UMBC frequency declined by 3.4% per year increase in age in SCD (95% CI: 2%, 4.7%, p < .001), but not in controls. A majority of children in all cohorts had an IgM concentration in the normal range for age and there were no differences between groups (p = .13). Subjects developed titers adequate for long-term protection to fewer serotypes in the polysaccharide vaccine than controls (14.7 vs. 19.4, p < .001). In this cohort, bacteremia was rare and specific clinical complications were not associated with UMBC proportion. In summary, UMBC deficiency occurs in SCD and is associated with age. Future studies should investigate B cell subsets prospectively and identify the mechanism of B cell loss in the spleen.

Pakikisama: Filipino Patient Perspectives on Health Care Access and Utilization.

PURPOSE: Filipinos have unique social determinants of health, cultural values, and beliefs that contribute to a higher prevalence of cardiovascular comorbidities such as hypertension, diabetes, and dyslipidemia. We aimed to identify Filipino values, practices, and belief systems that influenced health care access and utilization. METHODS: We conducted 1-on-1 semistructured interviews with self-identified Filipino patients. Our qualitative study utilized a constant-comparative approach for data collection, thematic coding, and interpretive analysis. RESULTS: We interviewed 20 Filipinos in a remote rural community to assess structural and social challenges experienced when interacting with the health care system. Our results suggest that Filipinos regard culture and language as pillars of health access. Filipinos trust clinicians who exhibited positive tone and body language as well as relatable and understandable communication. These traits are features of Pakikisama, a Filipino trait/value of "comfortableness and getting along with others." Relatability and intercultural values familiarity increased Filipino trust in a health care clinician. Filipinos may lack understanding about how to navigate the US Health care system, which can dissuade access to care. CONCLUSIONS: For the Filipino community, culture and language are fundamental components of health access. Health care systems have the opportunity to both improve intercultural clinical training and increase representation among clinicians and support staff to improve care delivery and navigation of health services. Participants reported not routinely relying on health care navigators.

An example of ventilatory limitation during cardiopulmonary exercise testing in a patient with COPD.

A 64-year-old obese gentleman attended for further evaluation of ongoing dyspnoea in the context of a previous diagnosis of moderate COPD treated with dual long-acting bronchodilators. A cardiopulmonary exercise test (CPET) was performed, which demonstrated reduced peak work and oxygen consumption with evidence of dynamic hyperinflation, abnormal gas exchange and ventilatory limitation despite cardiac reserve. The CPET clarified the physiological process underpinning the patient's dyspnoea and limiting the patient's activities. This, in turn, helped the clinician tailor the patient's management plan.

Generalizability of Tau and Amyloid Plasma Biomarkers in Alzheimer's Disease Cohorts of Diverse Genetic Ancestries.

Introduction: Plasma phosphorylated threonine-181 of Tau and amyloid beta are biomarkers for differential diagnosis and preclinical detection of Alzheimer disease (AD). Given differences in AD risk across diverse populations, generalizability of existing biomarker data is not assured. Methods: In 2,086 individuals of diverse genetic ancestries (African American, Caribbean Hispanic, and Peruvians) we measured plasma pTau-181 and Aß42/Aß40. Differences in biomarkers between cohorts and clinical diagnosis groups and the potential discriminative performance of the two biomarkers were assessed. Results: pTau-181 and Aß42/Aß40 were consistent across cohorts. Higher levels of pTau181 were associated with AD while Aß42/Aß40 had minimal differences. Correspondingly, pTau-181 had greater predictive value than Aß42/Aß40, however, the area under the curve differed between cohorts. Discussion: pTau-181 as a plasma biomarker for clinical AD is generalizable across genetic ancestries, but predictive value may differ. Combining genomic and biomarker data from diverse individuals will increase understanding of genetic risk and refine clinical diagnoses.

Viral Kinetics Model of SARS-CoV-2 Infection Informs Drug Discovery, Clinical Dose, and Regimen Selection.

Quantitative systems pharmacology (QSP) has been an important tool to project safety and efficacy of novel or repurposed therapies for the SARS-CoV-2 virus. Here, we present a QSP modeling framework to predict response to antiviral therapeutics with three mechanisms of action (MoA): cell entry inhibitors, anti-replicatives, and neutralizing biologics. We parameterized three distinct model structures describing virus-host interaction by fitting to published viral kinetics data of untreated COVID-19 patients. The models were used to test theoretical behaviors and map therapeutic design criteria of the different MoAs, identifying the most rapid and robust antiviral activity from neutralizing biologic and anti-replicative MoAs. We found good agreement between model predictions and clinical viral load reduction observed with anti-replicative nirmatrelvir/ritonavir (Paxlovid®) and neutralizing biologics bamlanivimab and casirivimab/imdevimab (REGEN-COV®), building confidence in the modeling framework to inform a dose selection. Finally, the model was applied to predict antiviral response with ensovibep, a novel DARPin therapeutic designed as a neutralizing biologic. We developed a new in silico measure of antiviral activity, area under the curve (AUC) of free spike protein concentration, as a metric with larger dynamic range than viral load reduction. By benchmarking to bamlanivimab predictions, we justified dose levels of 75, 225, and 600 mg ensovibep to be administered intravenously in a Phase 2 clinical investigation. Upon trial completion, we found model predictions to be in good agreement with the observed patient data. These results demonstrate the utility of this modeling framework to guide the development of novel antiviral therapeutics.

Dissection of core promoter syntax through single nucleotide resolution modeling of transcription initiation.

Our understanding of how the DNA sequences of cis-regulatory elements encode transcription initiation patterns remains limited. Here we introduce CLIPNET, a deep learning model trained on population-scale PRO-cap data that accurately predicts the position and quantity of transcription initiation with single nucleotide resolution from DNA sequence. Interpretation of CLIPNET revealed a complex regulatory syntax consisting of DNA-protein interactions in five major positions between -200 and +50 bp relative to the transcription start site, as well as more subtle positional preferences among different transcriptional activators. Transcriptional activator and core promoter motifs occupy different positions and play distinct roles in regulating initiation, with the former driving initiation quantity and the latter initiation position. We identified core promoter motifs that explain initiation patterns in the majority of promoters and enhancers, including DPR motifs and AT-rich TBP binding sequences in TATA-less promoters. Our results provide insights into the sequence architecture governing transcription initiation.

Factors associated with increased length of stay in degenerative cervical spine surgery: a cohort analysis from the Canadian Spine Outcomes and Research Network.

OBJECTIVE: Postoperative length of stay (LOS) significantly contributes to healthcare costs and resource utilization. The primary goal of this study was to identify patient, clinical, surgical, and institutional variables that influence LOS after elective surgery for degenerative conditions of the cervical spine. The secondary objectives were to examine the variability in LOS and institutional practices used to decrease LOS. METHODS: This was a multicenter observational retrospective cohort study of patients enrolled in the Canadian Spine Outcomes and Research Network (CSORN) between January 2015 and October 2020 who underwent elective anterior cervical discectomy and fusion (ACDF) (1-3 levels) or posterior cervical fusion (PCF) (between C2 and T2) with/without decompression for degenerative conditions of the cervical spine. Prolonged LOS was defined as LOS greater than the median for the ACDF and PCF populations. The principal investigators at each participating CSORN healthcare institution completed a survey to capture institutional practices implemented to reduce postoperative LOS. RESULTS: In total, 1228 patients were included (729 ACDF and 499 PCF patients). The median (IQR) LOS for ACDF and PCF were 1.0 (1.0) day and 5.0 (4.0) days, respectively. Predictors of prolonged LOS after ACDF were female sex, myelopathy diagnosis, lower baseline SF-12 mental component summary score, multilevel ACDF, and perioperative adverse events (AEs) (p < 0.05). Predictors of prolonged LOS after PCF were nonsmoking status, education less than high school, lower baseline numeric rating scale score for neck pain and EQ5D score, higher baseline Neck Disability Index score, and perioperative AEs (p < 0.05). Myelopathy did not significantly predict prolonged LOS within the PCF cohort after multivariate analysis. Of the 8 institutions (57.1%) with an enhanced recovery after surgery (ERAS) protocol or standardized protocol, only 3 reported using an ERAS protocol specific to patients undergoing ACDF or PCF. CONCLUSIONS: Patient and clinical factors predictive of prolonged LOS after ACDF and PCF are highly variable, warranting individual consideration for possible mitigation. Perioperative AEs remained a consistent independent predictor of prolonged LOS in both cohorts, highlighting the importance of preventing intra- and postoperative complications.

Pre-operative spine tumour embolization: Clinical outcomes and effect of embolization completeness.

INTRODUCTION: To assess the association between the impact of the completeness of pre-operative spine tumour embolisation and clinical outcomes, including estimated blood loss (EBL), neurological status and complications. METHODS: Retrospective chart review of all preoperative spine tumour embolisation procedures performed over 11 years by a single operator (2007-2018) at Vancouver General Hospital on 44 consecutive patients (mean age 57; 77% males) with 46 embolisation procedures, of which surgery was done en bloc in 26 cases and intralesional in the remaining 20. A multivariable negative binomial regression model was fit to examine the association between EBL and surgery type, tumour characteristics, embolisation completeness and operative duration. RESULTS: Among intralesional surgeries, complete versus incomplete embolisation was associated with reduced blood loss (772 vs 1428 mL, P < 0.01). There was no statistically significant difference in neurological outcomes or complications between groups. Highly vascular tumours correlated with greater blood loss than their less vascular counterparts, but tumour location did not have a statistically significant effect. CONCLUSION: This study provides evidence in support of our hypothesis that complete as opposed to incomplete tumour embolisation correlates with reduced blood loss in intralesional surgeries. Randomised control trials with larger samples are necessary to confirm this benefit and to ascertain other potential clinical benefits.

Association of leukemic molecular profile with efficacy of inotuzumab ozogamicin in adults with relapsed/refractory ALL.

The phase 3 INO-VATE trial demonstrated higher rates of remission, measurable residual disease negativity, and improved overall survival for patients with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) who received inotuzumab ozogamicin (InO) vs standard of care chemotherapy (SC). Here we examined associations between genomic alterations and the efficacy of InO. Of 326 randomized patients, 91 (InO, n=43; SC, n=48) had samples evaluable for genomic analysis. The spectrum of gene fusions and other genomic alterations observed was comparable with prior studies of adult ALL. Responses to InO were observed in all leukemic subtypes, genomic alterations, and risk groups. Significantly higher rates of complete remission (CR)/CR with incomplete count recovery rates were observed with InO vs SC in patients with BCR::ABL1-like ALL (85.7% [6/7] vs 0% [0/5] P=0.0076), with TP53 alterations (100% [5/5] vs 12.5% [1/8], P=0.0047), and in the high-risk BCR::ABL1- (BCR::ABL1-like, low hypodiploid, KMT2A-rearranged) group (83.3% [10/12] vs 10.5% [2/19]; P<0.0001). This retrospective, exploratory analysis of the INO-VATE trial demonstrated potential for benefit with InO for patients with R/R ALL across leukemic subtypes, including BCR::ABL1-like ALL, and for those bearing diverse genomic alterations. Further confirmation of the efficacy of InO in patients with R/R ALL exhibiting the BCR::ABL1-like subtype or harboring TP53 alterations is warranted. This trial was registered at www.clinicaltrials.gov as no. NCT01564784.

Dimeric peptoids as antibacterial agents.

Building upon our previous study on peptoid-based antibacterials which showed good activity against Gram-positive bacteria only, herein we report the synthesis of 34 dimeric peptoid compounds and the investigation of their activity against Gram-positive and Gram-negative pathogens. The newly designed peptoids feature a di-hydrophobic moiety incorporating phenyl, bromo-phenyl, and naphthyl groups, combined with variable lengths of cationic units such as amino and guanidine groups. The study also underscores the pivotal interplay between hydrophobicity and cationicity in optimizing efficacy against specific bacteria. The bromophenyl dimeric guanidinium peptoid compound 10j showed excellent activity against S. aureus 38 and E. coli K12 with MIC of 0.8 µg mL-1 and 6.2 µg mL-1, respectively. Further investigation into the mechanism of action revealed that the antibacterial effect might be attributed to the disruption of bacterial cell membranes, as suggested by tethered bilayer lipid membranes (tBLMs) and cytoplasmic membrane permeability studies. Notably, these promising antibacterial agents exhibited negligible toxicity against mammalian red blood cells. Additionally, the study explored the potential of 12 active compounds to disrupt established biofilms of S. aureus 38. The most effective biofilm disruptors were ethyl and octyl-naphthyl guanidinium peptoids (10c and 10 k). These compounds 10c and 10 k disrupted the established biofilms of S. aureus 38 with 51 % at 4x MIC (MIC = 17.6 µg mL-1 and 11.2 µg mL-1) and 56 %-58 % at 8x MIC (MIC = 35.2 µg mL-1 and 22.4 µg mL-1) respectively. Overall, this research contributes insights into the design principles of cationic dimeric peptoids and their antibacterial activity, with implications for the development of new antibacterial compounds.

Combining Scattering Experiments and Colloid Theory to Characterize Charge Effects in Concentrated Antibody Solutions.

Charges and their contribution to protein-protein interactions are essential for the key structural and dynamic properties of monoclonal antibody (mAb) solutions. In fact, they influence the apparent molecular weight, the static structure factor, the collective diffusion coefficient, or the relative viscosity, and their concentration dependence. Further, charges play an important role in the colloidal stability of mAbs. There exist standard experimental tools to characterize mAb net charges, such as the measurement of the electrophoretic mobility, the second virial coefficient, or the diffusion interaction parameter. However, the resulting values are difficult to directly relate to the actual overall net charge of the antibody and to theoretical predictions based on its known molecular structure. Here, we report the results of a systematic investigation of the solution properties of a charged IgG1 mAb as a function of concentration and ionic strength using a combination of electrophoretic measurements, static and dynamic light scattering, small-angle X-ray scattering, and tracer particle-based microrheology. We analyze and interpret the experimental results using established colloid theory and coarse-grained computer simulations. We discuss the potential and limits of colloidal models for the description of the interaction effects of charged mAbs, in particular pointing out the importance of incorporating shape and charge anisotropy when attempting to predict structural and dynamic solution properties at high concentrations.

Pre-operative expectations of patients with degenerative cervical myelopathy: an observational study from the Canadian Spine Outcomes and Research Network.

BACKGROUND: Despite an abundance of literature on degenerative cervical myelopathy (DCM), little is known about pre-operative expectations of these patients. PURPOSE: The primary objective was to describe patient pre-operative expectations. Secondary objectives included identifying patient characteristics associated with high pre-operative expectations and to determine if expectations varied depending on myelopathy severity. STUDY DESIGN: This was a retrospective study of a prospective multicenter, observational cohort of patients with DCM. PATIENT SAMPLE: Patients who consented to undergo surgical treatment between January 2019 and September 2022 were included. OUTCOMES MEASURES: An 11-domain expectation questionnaire was completed pre-operatively whereby patients quantified the expected change in each domain. METHODS: The most important expected change was captured. A standardized expectation score was calculated as the sum of each expectation divided by the maximal possible score. The high expectation group was defined by patients who had an expectation score above the 75th percentile. Predictors of patients with high expectations were determined using multivariable logistic regression models. RESULTS: There were 262 patients included. The most important patient expectation was preventing neurological worsening (40.8%) followed by improving balance when standing or walking (14.5%), improving independence in everyday activities (10.3%), and relieving arm tingling, burning and numbness (10%). Patients with mild myelopathy were more likely to select no worsening as the most important expected change compared to patients with severe myelopathy (p<.01). Predictors of high patient expectations were: having fewer comorbidities (OR -0.30 for every added comorbidity, 95% CI -0.59 to -0.10, p=.01), a shorter duration of symptoms (OR 0.92, 95% CI 0.35-1.19, p=.02), no contribution from "failure of other treatments" on the decision to undergo surgery (OR 1.49, 95% CI 0.56-2.71, p=.02) and more severe neck pain (OR 0.19 for 1 point increase, 95% CI 0.05-0.37, p=.01). CONCLUSIONS: Most patients undergoing surgery for DCM expect prevention of neurological decline, better functional status, and improvement in their myelopathic symptoms. Stopping neurological deterioration is the most important expected outcomes by patients.

A phase 1/2 study of pepinemab in children, adolescents, or young adults with recurrent or refractory solid tumors: A children's oncology group consortium report (ADVL1614).

PURPOSE: Pepinemab, a humanized IgG4 monoclonal antibody, targets the SEMA4D (CD100) antigen to inhibit binding to its high-affinity receptors (plexin B1/PLXNB1, plexin B2/PLXNB2) and low-affinity receptor (CD72). SEMA4D blockade leads to increased cytotoxic T-cell infiltration, delayed tumor growth, and durable tumor rejection in murine tumor models. Pepinemab was well tolerated and improved T cell infiltration in clinical studies in adults with refractory tumors. SEMA4D was identified as a strong candidate proto-oncogene in a model of osteosarcoma. Based on these preclinical and clinical data, we conducted a phase 1/2 study to determine the recommended phase 2 dose (RP2D), pharmacokinetics, pharmacodynamics, and immunogenicity, of pepinemab in pediatric patients with recurrent/refractory solid tumors, and activity in osteosarcoma. EXPERIMENTAL DESIGN: Pepinemab was administered intravenously on Days 1 and 15 of a 28-day cycle at 20 mg/kg, the adult RP2D. Part A (phase 1) used a Rolling 6 design; Part B (phase 2) used a Simon 2-stage design in patients with osteosarcoma. Pharmacokinetics and target saturation were evaluated in peripheral blood. RESULTS: Pepinemab (20 mg/kg) was well tolerated and no dose-limiting toxicities were observed during Part A. There were no objective responses. Two patients with osteosarcoma achieved disease control and prolonged stable disease. Pepinemab pharmacokinetics were similar to adults. CONCLUSIONS: Pepinemab (20 mg/kg) is safe, well tolerated and resulted in adequate and sustained target saturation in pediatric patients. Encouraging disease control in two patients with osteosarcoma warrants further investigation with novel combination strategies to modulate the tumor microenvironment and antitumor immune response. CLINICAL TRIAL REGISTRY: This trial is registered as NCT03320330 at Clinicaltrials.gov. DISCLAIMER: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

A lineage-specific STAT5BN642H mouse model to study NK-cell leukemia.

Patients with T- and NK-cell neoplasms frequently have somatic STAT5B gain-of-function mutations. The most frequent STAT5B mutation is STAT5BN642H, which is known to drive murine T-cell leukemia although its role in NK-cell malignancies is unclear. Introduction of the STAT5BN642H mutation into human NK-cell lines enhances their potential to induce leukemia in mice. We have generated a mouse model that enables tissue-specific expression of STAT5BN642H and have selectively expressed the mutated STAT5B in hematopoietic cells (N642Hvav/+) or exclusively in NK cells (N642HNK/NK). All N642Hvav/+ mice rapidly develop an aggressive T-/NK T-cell leukemia, whereas N642HNK/NK mice display an indolent NK-large granular lymphocytic leukemia (NK-LGLL) that progresses to an aggressive leukemia with age. Samples from NK-cell leukemia patients have a distinctive transcriptional signature driven by mutant STAT5B, which overlaps with that of murine leukemic N642HNK/NK NK cells. We have generated the first reliable STAT5BN642H-driven pre-clinical mouse model that displays an indolent NK-LGLL progressing to aggressive NK-cell leukemia. This novel in vivo tool will enable us to explore the transition from an indolent to an aggressive disease and will thus permit the study of prevention and treatment options for NK-cell malignancies.

A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma.

Recent studies suggest that long non-coding RNAs (lncRNAs) contribute to medulloblastoma (MB) formation and progression. We have identified an lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 (G3) MBs. lnc-HLX-2-7 RNA specifically accumulates in the promoter region of HLX, a sense-overlapping gene of lnc-HLX-2-7, which activates HLX expression by recruiting multiple factors, including enhancer elements. RNA sequencing and chromatin immunoprecipitation reveal that HLX binds to and activates the promoters of several oncogenes, including TBX2, LIN9, HOXM1, and MYC. Intravenous treatment with cerium-oxide-nanoparticle-coated antisense oligonucleotides targeting lnc-HLX-2-7 (CNP-lnc-HLX-2-7) inhibits tumor growth by 40%-50% in an intracranial MB xenograft mouse model. Combining CNP-lnc-HLX-2-7 with standard-of-care cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared with CNP-lnc-HLX-2-7 monotherapy. Thus, the lnc-HLX-2-7-HLX-MYC axis is important for regulating G3 MB progression, providing a strong rationale for using lnc-HLX-2-7 as a therapeutic target for G3 MBs.

Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing.

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.

Processing of corn-based dog foods through pelleting, baking and extrusion and their effect on apparent total tract digestibility and colonic health of adult dogs.

Different food processing parameters may alter starch granule structure and its cooking degree. With lower thermomechanical energy, more resistant starch (RS) is retained in the food, which may benefit gastrointestinal (GI) health. The objective of this study was to determine the effect of food processing on dietary utilization and dog gut health. Experimental diets containing 56% corn as the sole starch source were produced through pelleting, baking, and extrusion and compared to a baked control diet in which the corn was replaced with dextrose. The extruded diet resulted in the highest level (P < 0.05) of in vitro starch cook and lowest RS, while baked was intermediate and pelleted had the lowest starch cook and highest RS. To evaluate the in vivo effects of these treatments, 12 dogs were adapted to foods for 9 d, and feces were collected for 5 d in a replicated 4 × 4 Latin square design. Feces were scored for consistency using an ordinal scale, and parametric data included apparent digestibility (ATTD), parameters indicative of gut health, and the microbial composition, which was centered log-ratio transformed before operational taxonomic unit (OTU) analyses. Fecal scores were analyzed by ordinal logistic regression, and parametric data were analyzed as mixed models. Overall ATTD was greater (P < 0.05) in extruded, followed by baked and pelleted. Dogs fed the control had osmotic diarrhea, whereas dogs fed the other treatments had mostly acceptable fecal scores, with extrusion leading to the best fecal quality. The control also led to high fecal pH and low SCFAs, indicating dysbiosis. All corn foods had similar (P > 0.05) fecal SCFAs and extruded tended (P = 0.055) to promote higher fecal butyrate than baked and pelleted. The microbiome of dogs fed the corn foods had similar α diversity indices, and OTUs at the species and phyla levels were mostly alike and different from the control. In conclusion, the higher levels of in vitro RS did not translate into a better in vivo fermentation profile, and extruded kibble performed best regarding fecal quality, ATTD, and fecal SCFAs.

Dog foods were produced via extrusion, baking, and pelleting to yield increasing amounts of starch resistant to digestion (resistant starch [RS]). The foods were compared to a negative dextrose control that contained dextrose in place of starch. Amounts of cooked starch and RS were confirmed by in vitro methodologies. These foods were fed to healthy adult dogs in a Latin square design. Feces were scored for quality and collected to calculate apparent digestibility. Fresh feces were also collected for fecal short-chain fatty acids and microbiome evaluations. The corn-based extruded kibble was more digestible, followed by the baked and pelleted treatments. The extruded treatment produced stools closest to the ideal, but dogs fed the pelleted and baked also produced acceptable feces. The SCFA composition in the feces of dogs fed extruded was like the pelleted treatment, with a higher tendency to produce butyrate. Changes in fermentation were not a consequence of differences in microbiome composition among dogs fed corn-based foods. Dogs fed the control had osmotic diarrhea, with a higher fecal pH and higher proportions of branched-chain fatty acids, which was undesirable. The extruded food performed better overall than baked and pelleted, but they were all acceptable as food forms for dogs.

HtrA, fatty acids, and membrane protein interplay in Chlamydia trachomatis to impact stress response and trigger early cellular exit.

Chlamydia trachomatis is an intracellular bacterial pathogen that undergoes a biphasic developmental cycle, consisting of intracellular reticulate bodies and extracellular infectious elementary bodies. A conserved bacterial protease, HtrA, was shown previously to be essential for Chlamydia during the reticulate body phase, using a novel inhibitor (JO146). In this study, isolates selected for the survival of JO146 treatment were found to have polymorphisms in the acyl-acyl carrier protein synthetase gene (aasC). AasC encodes the enzyme responsible for activating fatty acids from the host cell or synthesis to be incorporated into lipid bilayers. The isolates had distinct lipidomes with varied fatty acid compositions. A reduction in the lipid compositions that HtrA prefers to bind to was detected, yet HtrA and MOMP (a key outer membrane protein) were present at higher levels in the variants. Reduced progeny production and an earlier cellular exit were observed. Transcriptome analysis identified that multiple genes were downregulated in the variants especially stress and DNA processing factors. Here, we have shown that the fatty acid composition of chlamydial lipids, HtrA, and membrane proteins interplay and, when disrupted, impact chlamydial stress response that could trigger early cellular exit. IMPORTANCE: Chlamydia trachomatis is an important obligate intracellular pathogen that has a unique biphasic developmental cycle. HtrA is an essential stress or virulence protease in many bacteria, with many different functions. Previously, we demonstrated that HtrA is critical for Chlamydia using a novel inhibitor. In the present study, we characterized genetic variants of Chlamydia trachomatis with reduced susceptibility to the HtrA inhibitor. The variants were changed in membrane fatty acid composition, outer membrane proteins, and transcription of stress genes. Earlier and more synchronous cellular exit was observed. Combined, this links stress response to fatty acids, membrane proteins, and HtrA interplay with the outcome of disrupted timing of chlamydial cellular exit.