BiliQML: A supervised machine-learning model to quantify biliary forms from digitized whole-slide liver histopathological images.

The progress of research focused on cholangiocytes and the biliary tree during development and following injury is hindered by limited available quantitative methodologies. Current techniques include two-dimensional standard histological cell-counting approaches, which are rapidly performed error-prone and lack architectural context; or three-dimensional analysis of the biliary tree in opacified livers, which introduce technical issues along with minimal quantitation. The present study aims to fill these quantitative gaps with a supervised machine learning model (BiliQML) able to quantify biliary forms in the liver of anti-Keratin 19 antibody-stained whole slide images. Training utilized 5,019 researcher-labeled biliary forms, which following feature selection, and algorithm optimization, generated an F-score of 0.87. Application of BiliQML on seven separate cholangiopathy models; genetic (Afp-CRE;Pkd1l1null/Fl, Alb-CRE;Rbp-jkfl/fl, Albumin-CRE; ROSANICD), surgical (bile duct ligation), toxicological (3,5-diethoxycarbonyl-1,4-dihydrocollidine), and therapeutic (Cyp2c70-/- with ileal bile acid transporter inhibition), allowed for a means to validate the capabilities, and utility of this platform. The results from BiliQML quantification revealed biological and pathological differences across these seven diverse models indicate a highly sensitive, robust, and scalable methodology for the quantification of distinct biliary forms. BiliQML is the first comprehensive machine-learning platform for biliary form analysis, adding much needed morphologic context to standard immunofluorescence - based histology, and provides clinical and basic-science researchers a novel tool for the characterization of cholangiopathies.

Microsecond dynamics control the HIV-1 Envelope conformation.

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although remarkable progress has been made in understanding the structures of various Env conformations, microsecond timescale dynamics have not been studied experimentally. Here, we used time-resolved, temperature-jump small-angle x-ray scattering to monitor structural rearrangements in an HIV-1 Env SOSIP ectodomain construct with microsecond precision. In two distinct Env variants, we detected a transition that correlated with known Env structure rearrangements with a time constant in the hundreds of microseconds range. A previously unknown structural transition was also observed, which occurred with a time constant below 10 µs, and involved an order-to-disorder transition in the trimer apex. Using this information, we engineered an Env SOSIP construct that locks the trimer in the prefusion closed state by connecting adjacent protomers via disulfides. Our findings show that the microsecond timescale structural dynamics play an essential role in controlling the Env conformation with impacts on vaccine design.

A molecular dynamics study of cell-penetrating peptide transportan-10 (TP10): Binding, folding and insertion to transmembrane state in zwitterionic membrane.

Transportan 10 (TP10) is a 21-residue, cationic, α-helical cell-penetrating peptide that can be used as a delivery vector for various bioactive molecules. Based on recent confocal microscopy studies, it is believed that TP10 can translocate across neutral lipid membrane passively, possibly as a monomer, without the formation of permanent pore. Here, we performed extensive molecular dynamics (MD) simulations of TP10W (Y3W variant of TP10) to find the microscopic details of binding, folding and insertion of TP10W to transmembrane state in POPC bilayer. Binding study with CHARMM36 force field showed that TP10W initially binds to the membrane surface in unstructured configuration, but it spontaneously folds into α-helical conformation under the lipid head groups. Further insertion of TP10W, changing from a surface bound state to a vertically oriented transmembrane state, was investigated via umbrella simulations. The resulting free energy profile shows a relatively small barrier between two states, suggesting a possible translocation pathway as a monomer. In fact, unbiased simulation of transmembrane TP10W revealed how a charged Lys side chain can move from one leaflet to the other without a significant free energy cost. Finally, we compared the results of TP10W simulations with those of point mutated variants (TP10W-K12A18 and TP10W-K19L) to understand the effect of charge distribution on the peptide. It was observed that such a conservative mutation can cause noticeable changes in the conformations of both surface bound and transmembrane states. The results of present study will be discussed in relation to the experimentally observed activities of TP10W against neutral membrane.

The Impact of Acupuncture on Self-Perceived Stress and ADHD Core Symptomatology in an Adult, Atomoxetine-taking ADHD Participant. Insights from an In-depth Single Case Study.

Background: Attention Deficit Hyperactivity Disorder (ADHD) includes inattention, hyperactivity, and impulsivity as core symptoms and is associated with increased self-perceived stress. Primary Study Objective: This article evaluates the impact of acupuncture (provided in addition to regular pharmacological treatment with Atomoxetine) on self-perceived stress and ADHD core symptomatology, compared to atomoxetine (ATX) alone. Methods/Design: In-depth single case study, involving a mixed methods approach with questionnaires and interviews was used. The participant completed two rating scales. Additionally, semi-structured interviews were held. Qualitative data were subjected to content analysis and both sets of data were triangulated. Setting: Data collection/intervention (treatments) took place at an Acupuncture clinic in Hamburg, Germany, EU. Participants: One adult, atomoxetine-taking ADHD participant. Intervention: Acupuncture according to Chinese medicine-diagnosis twice/week, over the course of eight weeks, following a pre-defined but flexible point protocol. Primary Outcome Measures: 1) The Current Symptom Scale (CSS) and the Perceived Stress Scale (PSS). 2) Semi-structured interviews. Results: Acupuncture treatments in addition to regular ATX intake positively affected the participant's ADHD symptoms (PSS - 31%, total score of the CSS - 47%). There was a considerable decrease in subgroup scores (attention deficit - 39%; functionality impairment - 55%; hyperactivity/impulsivity - 53%; impulsivity - 30%). Post-interventional interviews showed perceived increased self-control, (self-) awareness and centeredness. Combined treatment was perceived as more beneficial than pharmaceutical treatment alone. Conclusion: Acupuncture treatment appears to have a positive impact on both self-perception of stress and ADHD core symptomatology. Findings were partially congruent with the reviewed research literature but due to limitations/risks of bias (ROBs) associated with the design, no concrete conclusions regarding a potential method-related specificity can be drawn. Further research with larger samples and a more robust design is recommended.

Microsecond dynamics control the HIV-1 envelope conformation.

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although significant progress has been made in understanding the structures of various Env conformations and transition intermediates that occur within the millisecond timescale, faster transitions in the microsecond timescale have not yet been observed. In this study, we employed time-resolved, temperature-jump small angle X-ray scattering to monitor structural rearrangements in an HIV-1 Env ectodomain construct with microsecond precision. We detected a transition correlated with Env opening that occurs in the hundreds of microseconds range and another more rapid transition that preceded this opening. Model fitting indicated that the early rapid transition involved an order-to-disorder transition in the trimer apex loop contacts, suggesting that conventional conformation-locking design strategies that target the allosteric machinery may be ineffective in preventing this movement. Utilizing this information, we engineered an envelope that locks the apex loop contacts to the adjacent protomer. This modification resulted in significant angle-of-approach shifts in the interaction of a neutralizing antibody. Our findings imply that blocking the intermediate state could be crucial for inducing antibodies with the appropriate bound state orientation through vaccination.

Liver-restricted deletion of the biliary atresia candidate gene Pkd1l1 causes bile duct dysmorphogenesis and ciliopathy.

BACKGROUND AND AIMS: A recent multicenter genetic exploration of the biliary atresia splenic malformation syndrome identified mutations in the ciliary gene PKD1L1 as candidate etiologic contributors. We hypothesized that deletion of Pkd1l1 in developing hepatoblasts would lead to cholangiopathy in mice. APPROACH AND RESULTS: CRISPR-based genome editing inserted loxP sites flanking exon 8 of the murine Pkd1l1 gene. Pkd1l1Fl/Fl cross-bred with alpha-fetoprotein-Cre expressing mice to generate a liver-specific intrahepatic Pkd1l1 -deficient model (LKO). From embryonic day 18 through week 30, control ( Fl/Fl ) and LKO mice were evaluated with standard serum chemistries and liver histology. At select ages, tissues were analyzed using RNA sequencing, immunofluorescence, and electron microscopy with a focus on biliary structures, peribiliary inflammation, and fibrosis. Bile duct ligation for 5 days of Fl/Fl and LKO mice was followed by standard serum and liver analytics. Histological analyses from perinatal ages revealed delayed biliary maturation and reduced primary cilia, with progressive cholangiocyte proliferation, peribiliary fibroinflammation, and arterial hypertrophy evident in 7- to 16-week-old LKO versus Fl/Fl livers. Following bile duct ligation, cholangiocyte proliferation, peribiliary fibroinflammation, and necrosis were increased in LKO compared with Fl/Fl livers. CONCLUSIONS: Bile duct ligation of the Pkd1l1 -deficient mouse model mirrors several aspects of the intrahepatic pathophysiology of biliary atresia in humans including bile duct dysmorphogenesis, peribiliary fibroinflammation, hepatic arteriopathy, and ciliopathy. This first genetically linked model of biliary atresia, the Pkd1l1 LKO mouse, may allow researchers a means to develop a deeper understanding of the pathophysiology of this serious and perplexing disorder, including the opportunity to identify rational therapeutic targets.

Prior immunization against an intracellular antigen enhances subsequent red blood cell alloimmunization in mice.

Antibodies against red blood cell (RBC) alloantigens can increase morbidity and mortality among transfusion recipients. However, alloimmunization rates can vary dramatically, as some patients never generate alloantibodies after transfusion, whereas others not only become alloimmunized but may also be prone to generating additional alloantibodies after subsequent transfusion. Previous studies suggested that CD4 T-cell responses that drive alloantibody formation recognize the same alloantigen engaged by B cells. However, because RBCs express numerous antigens, both internally and externally, it is possible that CD4 T-cell responses directed against intracellular antigens may facilitate subsequent alloimmunization against a surface RBC antigen. Here, we show that B cells can acquire intracellular antigens from RBCs. Using a mouse model of donor RBCs expressing 2 distinct alloantigens, we demonstrate that immune priming to an intracellular antigen, which would not be detected by any currently used RBC compatibility assays, can directly influence alloantibody formation after exposure to a subsequent distinct surface RBC alloantigen. These findings suggest a previously underappreciated mechanism whereby transfusion recipient responders may exhibit an increased rate of alloimmunization because of prior immune priming toward intracellular antigens.

Doxorubicin for treatment of histiocytic sarcoma in dogs: 31 cases (2003-2017).

OBJECTIVE: To evaluate the efficacy of doxorubicin for treatment of histiocytic sarcoma (HS) in dogs, whether administered as the sole treatment or as an adjunct to surgery or radiation therapy. ANIMALS: 31 client-owned dogs with localized or disseminated HS examined between 2003 and 2017. PROCEDURES: Medical records were reviewed retrospectively, and data were collected. The Kaplan-Meier method was used to estimate time-to-progression from the date of first doxorubicin administration and survival time from initial diagnosis. Factors that could be associated with poorer outcomes with doxorubicin treatment were analyzed with log-rank tests. RESULTS: The objective response rate (ORR) was 26%. When stratified by disease status, dogs with localized and disseminated forms experienced 43% and 21% ORRs, respectively. Median time to progression after initiating doxorubicin treatment (n = 30 dogs) was 42 days. Median survival time from initial diagnosis to death (n = 29 dogs) was 169 days. Complete responses were obtained in only 2 dogs that had localized disease and received multimodality therapy. CLINICAL RELEVANCE: Benefits of doxorubicin administration in canine HS are modest, with a limited ORR and delay in tumor progression, and are comparable to effects attained with other single-agent regimens.

Ileal bile acid transporter inhibition in Cyp2c70 KO mice ameliorates cholestatic liver injury.

Cyp2c70 is the liver enzyme in rodents responsible for synthesis of the primary 6-hydroxylated muricholate bile acid (BA) species. Cyp2c70 KO mice are devoid of protective, hydrophilic muricholic acids, leading to a more human-like BA composition and subsequent cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be therapeutic in cholestatic models. Here, we aimed to determine if IBAT inhibition with SC-435 is protective in Cyp2c70 KO mice. As compared to WT mice, we found male and female Cyp2c70 KO mice exhibited increased levels of serum liver injury markers, and our evaluation of liver histology revealed increased hepatic inflammation, macrophage infiltration, and biliary cell proliferation. We demonstrate serum and histologic markers of liver damage were markedly reduced with SC-435 treatment. Additionally, we show hepatic gene expression in pathways related to immune cell activation and inflammation were significantly upregulated in Cyp2c70 KO mice and reduced to levels indistinguishable from WT with IBAT inhibition. In Cyp2c70 KO mice, the liver BA content was significantly increased, enriched in chenodeoxycholic acid, and more hydrophobic, exhibiting a hydrophobicity index value and red blood cell lysis properties similar to human liver BAs. Furthermore, we determined IBAT inhibition reduced the total hepatic BA levels but did not affect overall hydrophobicity of the liver BAs. These findings suggest that there may be a threshold in the liver for pathological accretion of hydrophobic BAs and reducing hepatic BA accumulation can be sufficient to alleviate liver injury, independent of BA pool hydrophobicity.

Clodronate inhibits alloimmunization against distinct red blood cell alloantigens in mice.

BACKGROUND: Alloimmunization can be a significant barrier to red blood cell (RBC) transfusion. While alloantigen matching protocols hold promise in reducing alloantibody formation, transfusion-dependent patients can still experience RBC alloimmunization and associated complications even when matching protocols are employed. As a result, complementary strategies capable of actively preventing alloantibody formation following alloantigen exposure are warranted. STUDY DESIGN AND METHODS: We examined whether pharmacological removal of macrophages using clodronate may provide an additional strategy to actively inhibit RBC alloimmunization using two preclinical models of RBC alloimmunization. To accomplish this, mice were treated with clodronate, followed by transfusion of RBCs expressing the HOD (HEL, OVA, and Duffy) or KEL antigens. On days 5 and 14 post transfusion, anti-HOD or anti-KEL IgM and IgG antibodies were evaluated. RESULTS: Low dose clodronate effectively eliminated key marginal zone macrophage populations from the marginal sinus. Prior treatment with clodronate, but not empty liposomes, also significantly inhibited IgM and IgG anti-HOD alloantibody formation following transfusion of HOD RBCs. Similar exposure to clodronate inhibited IgM and IgG antibody formation following KEL RBC transfusion. CONCLUSIONS: Clodronate can inhibit anti-HOD and anti-KEL antibody formation following RBC transfusion in preclinical models. These results suggest that clodronate may provide an alternative approach to actively inhibit or prevent the development of alloantibodies following RBC transfusion, although future studies will certainly be needed to fully explore this possibility.

HIV-1 Envelope Conformation, Allostery, and Dynamics.

The HIV-1 envelope glycoprotein (Env) mediates host cell fusion and is the primary target for HIV-1 vaccine design. The Env undergoes a series of functionally important conformational rearrangements upon engagement of its host cell receptor, CD4. As the sole target for broadly neutralizing antibodies, our understanding of these transitions plays a critical role in vaccine immunogen design. Here, we review available experimental data interrogating the HIV-1 Env conformation and detail computational efforts aimed at delineating the series of conformational changes connecting these rearrangements. These studies have provided a structural mapping of prefusion closed, open, and transition intermediate structures, the allosteric elements controlling rearrangements, and state-to-state transition dynamics. The combination of these investigations and innovations in molecular modeling set the stage for advanced studies examining rearrangements at greater spatial and temporal resolution.

The Impact of the Tertiary Survey in an Established Trauma Program.

INTRODUCTION: The trauma tertiary survey (TTS) was first described in 1990 and is recognized as an essential practice in trauma care. The TTS remains effective in detecting secondary injuries in the modern era. METHODS: Trauma patients discharged between August 1, 2016, and December 31, 2016, were identified in our trauma registry. Collected data include TTS completion rates, detection of injuries, type of provider, and timing. TTS documentation was qualitatively evaluated. RESULTS: Out of 407 patients, 264 patients (65%) received a TTS. Injury detection rate was 1.1.%. Average time to TTS was 41 hours. TTS were completed by resident physicians (46%) and advanced practice providers (APPs; 46%). TTS documentation was more complete for APPs than for resident physicians. CONCLUSION: TTS remains an integral component of modern trauma care. Ongoing education on the significance of TTS and the importance of thorough documentation is essential. Provision of real-time feedback to providers is also critical for improving current practices.

Rectal foreign bodies: national outcomes after the operating room.

PURPOSE: Rectal foreign bodies can pose a unique problem to the acute care surgeon or emergency room physician. Little data exists on the patient with such a diagnosis, outside of case reports, and institutional cohorts. This study describes demographics and outcomes for this patient population. METHODS: An analysis of the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database (2015 to 2018) was performed, capturing patients with a postoperative diagnosis of foreign body of the anus or rectum. Patients were stratified into two groups: those who underwent removal under anesthesia (EUA) and those who required surgical removal. Baseline demographics and outcomes were compared and described. RESULTS: A total of 109 patients were included in the study. The average age was 49.29 ± 14.63 years. The majority of the population was male (92.66%) and white (78.90%). Demographics and preoperative variables were clinically similar between patients receiving an EUA or an operation. Length of stay was longer in patients receiving an operation (4.84 ± 3.27 versus 1.39 ± 3.20 days in those receiving an EUA, p < 0.0001). About 97.24% of the population discharged to their home residence. CONCLUSIONS: This study describes a population of patients admitted for retained foreign body of the rectum, in a population larger than that can be described in a typical institutional review. EUA can be a safe approach to foreign body removal, with laparotomy carrying the typical risks of surgery. This population requires further study to identify the mechanisms and risk factors for alimentary tract injury to reduce operative interventions and improve outcomes.

Ketoacidosis in Euglycemic Patients With Type 2 Diabetes After Abdominal Surgery.

Diabetic ketoacidosis is defined as hyperglycemia >250 mg/dL with metabolic acidosis of arterial pH <7.3, serum bicarbonate <18 mEq/L with positive urine and serum ketones and an anion gap >10. Euglycemic ketoacidosis has been reported in patients with type 2 diabetes and in patients with type 1 diabetes. However, as a surgical complication, euglycemic ketoacidosis has not been reported. We report 2 cases from 2 teaching tertiary care centers of patients with type 2 diabetes who developed high-gap ketoacidosis in an intensive care unit while recovering from emergent abdominal surgery. Both patients developed altered mental status, metabolic acidosis with a bicarbonate level as low as 14 mEq/L, and an anion gap > 18, without hyperglycemia. Both patients had ß-hydroxybutyrate levels > 5 mmol/L.

Anemia induces gut inflammation and injury in an animal model of preterm infants.

BACKGROUND: While very low birth weight (VLBW) infants often require multiple red blood cell transfusions, efforts to minimize transfusion-associated risks have resulted in more restrictive neonatal transfusion practices. However, whether restrictive transfusion strategies limit transfusions without increasing morbidity and mortality in this population remains unclear. Recent epidemiologic studies suggest that severe anemia may be an important risk factor for the development of necrotizing enterocolitis (NEC). However, the mechanism whereby anemia may lead to NEC remains unknown. STUDY DESIGN AND METHODS: The potential impact of anemia on neonatal inflammation and intestinal barrier disruption, two well-characterized predisposing features of NEC, was defined by correlation of hemoglobin values to cytokine levels in premature infants and by direct evaluation of intestinal hypoxia, inflammation and gut barrier disruption using a pre-clinical neonatal murine model of phlebotomy-induced anemia (PIA). RESULTS: Increasing severity of anemia in the preterm infant correlated with the level of IFN-gamma, a key pro-inflammatory cytokine that may predispose an infant to NEC. Gradual induction of PIA in a pre-clinical model resulted in significant hypoxia throughout the intestinal mucosa, including areas where intestinal macrophages reside. PIA-induced hypoxia significantly increased macrophage pro-inflammatory cytokine levels, while reducing tight junction protein ZO-1 expression and increasing intestinal barrier permeability. Macrophage depletion reversed the impact of anemia on intestinal ZO-1 expression and barrier function. CONCLUSIONS: Taken together, these results suggest that anemia can increase intestinal inflammation and barrier disruption likely through altered macrophage function, leading to the type of predisposing intestinal injury that may increase the risk for NEC.

Landscape and local site variables differentially influence pollinators and pollination services in urban agricultural sites.

Urbanization has detrimental effects on biodiversity and ecosystem functioning, as agricultural and semi-natural habitats are converted into landscapes dominated by built features. Urban agricultural sites are a growing component of urban landscapes and have potential to serve as a source of biodiversity conservation and ecosystem service provisioning in urban areas. In 19 urban agricultural sites, we investigated how surrounding land cover and local site variables supported bees and pollination services. We found the abundance of bees differentially responded to landscape and local scale variables depending on body size and nesting habit. Large-bodied bees, Bombus and Apis species, were positively associated with increasing amounts of impervious cover, while the abundance of small-bodied soil nesting Halictus species increased as the proportion of flower area, a local variable, increased. Bee richness declined with increasing levels of impervious cover, while bee community composition changed along a gradient of increasing impervious cover. Pollination services, measured at each site using sentinel cucumber plants, declined as hardscape, a local variable, increased. To improve bee conservation and pollination services in urban agricultural sites, our results suggest urban planning strategies should minimize impervious cover at large spatial scales while land managers should focus locally on incorporating floral resources, which increases food and nesting resources especially for smaller bee species. Local site design coupled with regional urban planning can advance the success of urban agriculture, while benefiting biodiversity by creating opportunities for pollinator conservation in urban landscapes.

Harvesting effects on wild bee communities in bioenergy grasslands depend on nesting guild.

Conversion of annual crops to native perennial grasslands for bioenergy production may help conserve wild bees by enhancing nest and food resources. However, bee response to the disturbance of biomass harvesting may depend on their nesting location, thus their vulnerability to nest destruction, and the response of the forb community on which they forage. Moreover, because bees have long foraging ranges, effects of local harvesting may depend on the amount of natural habitat in the surrounding landscape. We performed a large-scale one- and two-year experiment in Michigan and Wisconsin, USA, respectively, to examine how grassland harvesting, landscape context, and study year affect the forb community, above- and belowground-nesting bee species richness, community composition, trap nest emergence, and visitation rate. In Wisconsin, harvesting increased forb richness, cover, and evenness compared to unharvested control sites. Harvesting negatively affected aboveground-nesting bee richness and emergence from trap nests, possibly because of nest destruction during the previous harvest. By contrast, harvesting positively affected belowground-nesting bee richness, possibly because of the greater food resource availability and reduced thatch allowing greater access to nesting sites in the soil. Harvesting also affected bee community composition, reflecting the increase in belowground-nesting species at harvested sites. Despite harvesting effects on forb and bee communities, there was no effect on flower visitation rate, indicating little effect on pollination function. We did not find a harvest by landscape context interaction, which, in combination with the negative harvesting effect on trap nest emergence, suggests that harvesting can affect local population growth rather than simply affecting forager aggregation in different resource environments. For bees, there was no harvest by study year interaction, indicating a consistent response over a short timescale. Similarly, in Michigan, belowground-nesting species also responded positively to harvesting, which was more pronounced in sandier soils that are preferred for nesting. However, other components of the Michigan bee and forb communities were not significantly affected by biomass harvesting. Overall, our study demonstrates that harvesting grasslands can positively affect the ~80% of bee species that nest belowground by enhancing nest and/or forage resources, but that conserving aboveground nesters may require leaving some area unharvested.