Microinstrumentation for Brain Organoids.

Brain organoids are three-dimensional aggregates of self-organized differentiated stem cells that mimic the structure and function of human brain regions. Organoids bridge the gaps between conventional drug screening models such as planar mammalian cell culture, animal studies, and clinical trials. They can revolutionize the fields of developmental biology, neuroscience, toxicology, and computer engineering. Conventional microinstrumentation for conventional cellular engineering, such as planar microfluidic chips; microelectrode arrays (MEAs); and optical, magnetic, and acoustic techniques, has limitations when applied to three-dimensional (3D) organoids, primarily due to their limits with inherently two-dimensional geometry and interfacing. Hence, there is an urgent need to develop new instrumentation compatible with live cell culture techniques and with scalable 3D formats relevant to organoids. This review discusses conventional planar approaches and emerging 3D microinstrumentation necessary for advanced organoid-machine interfaces. Specifically, this article surveys recently developed microinstrumentation, including 3D printed and curved microfluidics, 3D and fast-scan optical techniques, buckling and self-folding MEAs, 3D interfaces for electrochemical measurements, and 3D spatially controllable magnetic and acoustic technologies relevant to two-way information transfer with brain organoids. This article highlights key challenges that must be addressed for robust organoid culture and reliable 3D spatiotemporal information transfer.

Characterizing Persons With HIV/HCV Coinfection Who Remain Untreated for Hepatitis C at Four HIV Clinics in Connecticut (CT): Role of Multiple Overlapping Barriers at the Individual and Clinic System Levels.

Introduction. Direct-acting antiviral medications have made hepatitis C virus (HCV) cure possible for >95% of persons with chronic HCV infection, including those coinfected with HIV. Achieving strategic HCV elimination targets requires an understanding of system, provider, and patient-level barriers to treatment. We explored such barriers among persons with HIV/HCV coinfection who remained untreated for HCV. Methods. Among four primary care HIV clinics in CT with high rates of HCV cure, 25 patients with HIV/HCV coinfection were eligible (no HCV treatment as of March 31, 2021). We conducted retrospective chart reviews of demographics, clinical practice patterns, patient-specific issues such as housing, transportation, food security, and presence of mental health and substance use problems. Results. Among untreated patients, 13 (51%) were female; 17 (68%) were Black; median age was 62 years old. The majority (84%) had injecting drug use (IDU) as HIV transmission risk factor; 14 (56%) were prescribed medication-assisted treatment. Median time since HIV and HCV diagnosis was 25 and 19 years, respectively. Clinic-level barriers were noted in 19 (76%) and included lack of evaluation, treatment not recommended or implemented. Concomitant structural barriers included unstable housing for 11 (44%) and lack of transportation for eight (32%). Most patients had history of illicit substance use (84%) and mental health issues (68%). Many (76%) had multiple potential barriers. Conclusions. Multiple overlapping barriers spanning clinic and patient level domains including social determinants of health were the norm in persons with long-standing HIV/HCV coinfection who have not received HCV treatment. Interventions will require innovative, multi-disciplinary and personalized approaches.

Management of Weight Regain Following Bariatric Surgery: Behavioral Intervention and Pharmacotherapy.

INTRODUCTION: Bariatric surgery is the most effective intervention currently available for significant and durable weight loss, but weight regain after surgery is not uncommon. This paper focuses on updates in behavioral interventions and pharmacotherapy to combat weight regain after bariatric surgery. AREAS COVERED: This paper critically reviews both prospective and retrospective studies assessing pharmacotherapy in post-bariatric surgical patients published within the past 5 years. It also evaluates updates in behavioral therapies and delivery of the therapies in this patient population. EXPERT OPINION: Weight regain after bariatric surgery is common. Patients who experience weight regain should be evaluated and treated by a multidisciplinary team. Antiobesity pharmacotherapy should be considered for those who qualify as an adjunct to lifestyle modifications, along with behavioral interventions such as cognitive behavioral therapy.

Longitudinal Improvements in Viral Suppression for Persons With New HIV Diagnosis Receiving Care in the Ryan White Program: A 10-Year Experience in New Haven, CT (2009-2018).

Background: The Ryan White (RW) program funds medical and other support services for low-income persons with HIV, significantly improving progress along the HIV care continuum. Although the program has shown overall improvements in achievement of viral suppression, the relative contributions of changes in clinical practice and RW service components to the optimization of the HIV care continuum, particularly for those with new HIV diagnoses, remain unknown. Methods: The target population was patients with recent HIV diagnoses who received care at RW-funded clinics in the greater New Haven area between 2009 and 2018. Client data were extracted from the RW-funded database, CAREWare, and the electronic medical record. Primary outcomes included time between HIV diagnosis and first HIV primary care (PC) visit, antiretroviral therapy (ART) initiation, and viral suppression (VS). Results: There were 386 eligible patients. Between 2009 and 2018, the median number of days from HIV diagnosis to first PC visit decreased from 58.5 to 8.5 days, and ART initiation decreased from 155 to 9 days. In 2018, 86% of participants achieved viral suppression within 1 year, compared with 2.5% in 2009. Patients who initiated single-tablet ART and integrase inhibitor-containing regimens were more likely to reach viral suppression within 1 year (P < .001). Receipt of medical case management services was also associated with achieving viral suppression (P < .001). Conclusions: Longitudinal improvements over 10 years in ART initiation and viral suppression were observed due to clinical advances and their effective implementation through the RW comprehensive care model. Further study of the essential components promoting these outcomes is needed.

Pharmacy-Based Interventions to Increase Use of HIV Pre-exposure Prophylaxis in the United States: A Scoping Review.

HIV pre-exposure prophylaxis (PrEP) remains underutilized in the U.S. Since greater than 85% of PrEP prescriptions are filled at commercial pharmacies, pharmacists are uniquely positioned to increase PrEP use. This scoping review explores pharmacy-based initiatives to increase PrEP use. We searched PubMed, PsycINFO, CINAHL, and Scopus for peer-reviewed studies on pharmacist-led interventions to increase PrEP use or pharmacy-based PrEP initiatives. Forty-nine articles were included in this review. Overall, studies demonstrated that patients expressed strong support for pharmacist prescription of PrEP. Three intervention designs compared changes in PrEP initiation or knowledge pre- and post-intervention. Commentary/review studies recommended PrEP training for pharmacists, policy changes to support pharmacist screening for HIV and PrEP prescription, and telemedicine to increase prescriptions. Pharmacists could play key roles in improving PrEP use in the U.S. Studies that assess improvements in PrEP use after interventions such as PrEP prescription, PrEP-specific training, and adherence monitoring by pharmacists are needed.

SENP3 Promotes an Mff-Primed Bcl-x L -Drp1 Interaction Involved in Cell Death Following Ischemia.

Dysregulation of the mitochondrial fission machinery has been linked to cell death following ischemia. Fission is largely dependent on recruitment of Dynamin-related protein 1 (Drp1) to the receptor Mitochondrial fission factor (Mff) located on the mitochondrial outer membrane (MOM). Drp1 is a target for SUMOylation and its deSUMOylation, mediated by the SUMO protease SENP3, enhances the Drp1-Mff interaction to promote cell death in an oxygen/glucose deprivation (OGD) model of ischemia. Another interacting partner for Drp1 is the Bcl-2 family member Bcl-x L , an important protein in cell death and survival pathways. Here we demonstrate that preventing Drp1 SUMOylation by mutating its SUMO target lysines enhances the Drp1-Bcl-x L interaction in vivo and in vitro. Moreover, SENP3-mediated deSUMOylation of Drp1 promotes the Drp1-Bcl-x L interaction. Our data suggest that Mff primes Drp1 binding to Bcl-x L at the mitochondria and that Mff and Bcl-x L can interact directly, independent of Drp1, through their transmembrane domains. Importantly, SENP3 loss in cells subjected to OGD correlates with reduced Drp1-Bcl-x L interaction, whilst recovery of SENP3 levels in cells subjected to reoxygenation following OGD correlates with increased Drp1-Bcl-x L interaction. Expressing a Bcl-x L mutant with defective Drp1 binding reduces OGD plus reoxygenation-evoked cell death. Taken together, our results indicate that SENP3-mediated deSUMOlyation promotes an Mff-primed Drp1-Bcl-x L interaction that contributes to cell death following ischemia.

A combined modelling and experimental study of heat shock factor SUMOylation in response to heat shock.

One of the most important questions in cell biology is how cell fate is determined when exposed to extreme stresses such as heat shock. It has been long understood that organisms exposed to high temperature stresses typically protect themselves with a heat shock response (HSR), where accumulation of denatured or unfolded proteins triggers the synthesis of heat shock proteins (HSPs) through the heat shock transcription factor, e.g., heat shock factor 1 (HSF1). In this study, a dynamical model validated with experiments is presented to analyse the role of HSF1 SUMOylation in response to heat shock. Key features of this model are inclusion of heat shock response and SUMOylation of HSF1, and HSP synthesis at molecular level, describing the dynamical evolution of the key variables involved in the regulation of HSPs. The model has been employed to predict the SUMOylation levels of HSF1 with different external temperature stimuli. The results show that the SUMOylated HSF1 levels agree closely with the experimental findings. This demonstrates the validity of this nonlinear dynamic model for the important role of SUMOylation in response to heat shock.

New Proteomic Signatures to Distinguish Between Zika and Dengue Infections.

Distinguishing between Zika and dengue virus infections is critical for accurate treatment, but we still lack detailed understanding of their impact on their host. To identify new protein signatures of the two infections, we used next-generation proteomics to profile 122 serum samples from 62 Zika and dengue patients. We quantified >500 proteins and identified 13 proteins that were significantly differentially expressed (adjusted p-value < 0.05). These proteins typically function in infection and wound healing, with several also linked to pregnancy and brain function. We successfully validated expression differences with Carbonic Anhydrase 2 in both the original and an independent sample set. Three of the differentially expressed proteins, i.e., Fibrinogen Alpha, Platelet Factor 4 Variant 1, and Pro-Platelet Basic Protein, predicted Zika virus infection at a ∼70% true-positive and 6% false-positive rate. Further, we showed that intraindividual temporal changes in protein signatures can disambiguate diagnoses and serve as indicators for past infections. Taken together, we demonstrate that serum proteomics can provide new resources that serve to distinguish between different viral infections.

Cabazitaxel schedules in metastatic castration-resistant prostate cancer: a review.

Cabazitaxel (25 mg/m2 every 3 weeks) is the standard second-line chemotherapy for patients with metastatic castration-resistant prostate cancer previously treated with docetaxel. It is associated with a risk of neutropenic complications, which may be a barrier to its use in daily clinical practice, particularly in frail elderly patients. Here the authors reviewed key studies conducted with cabazitaxel (TROPIC, PROSELICA, AFFINITY, CARD and the European compassionate use program) and pilot studies with adapted schedules. Based on this review, the use of prophylactic granulocyte colony-stimulating factor from cycle 1 appears crucial to maximize the benefit-risk ratio of cabazitaxel in metastatic castration-resistant prostate cancer. Preliminary data with alternative schedules look promising, especially for frail patients. Results of the ongoing Phase III CABASTY trial (ClinicalTrials.gov: NCT02961257) are awaited.

Toxicity of locoregional radiotherapy in combination with bevacizumab in patients with non-metastatic breast cancer (TOLERAB): Final long-term evaluation.

BACKGROUND AND PURPOSE: Few data are available concerning the safety of bevacizumab (B) in combination with locoregional radiation therapy (RT). The objective of this study was to evaluate the 5-year late toxicity of concurrent B and RT in non-metastatic breast cancer. MATERIALS AND METHODS: This multicentre prospective study included non-metastatic breast cancer patients enrolled in phase 3 clinical trials evaluating B with concurrent RT versus RT alone. All patients received neoadjuvant or adjuvant chemotherapy and normofractionated breast or chest wall RT, with or without regional lymph node RT. B was administered at an equivalent dose of 5 mg/kg once a week for 1 year. The safety profile was evaluated 1, 3 and 5 years after completion of radiotherapy. RESULTS: A total of 64 patients were included between November 2007 and April 2010. Median follow-up was 60 months (12-73) and 5-year late toxicity data were available for 46 patients. The majority of tumours were triple-negative (68.8%), tumour size <2cm (41.3%) with negative nodal status (50.8%). Median total dose of B was 15,000mg and median duration was 11.2 months. No grade ≥3 toxicity was observed. Only 8 patients experienced grade 1-2 toxicities: n = 3 (6.5%) grade 1 lymphedema, n = 2 (4.3%) grade 1 pain, n = 1 (2.2%) grade 2 lymphedema, n = 1 (2.2%) grade 1 fibrosis. Five-year overall survival was 93.8%, disease-free survival was 89% and locoregional recurrence-free survival was 93.1%. CONCLUSION: Concurrent B and locoregional RT are associated with acceptable 5-year toxicity in patients with non-metastatic breast cancer. No grade ≥3 toxicity was observed.

Safety and efficacy of 2-weekly cabazitaxel in metastatic castration-resistant prostate cancer.

OBJECTIVES: To evaluate the safety and efficacy of a 2-weekly cabazitaxel schedule in patients with metastatic castration-resistant prostate cancer (mCRPC). MATERIALS AND METHODS: During the period October 2013 to February 2016, 43 patients with mCRPC were treated with cabazitaxel (16 mg/m2 , on days 1 and 15 of a 4-week cycle) together with prophylactic granulocyte colony-stimulating factor (G-CSF). The safety profile and efficacy (prostate-specific antigen [PSA] response; biological, clinical or radiological progression-free survival [PFS] and overall survival [OS]) of the treatment were analysed. RESULTS: All patients had received prior docetaxel and 79.1% abiraterone acetate. At inclusion, 46.5% were aged >70 years and 27.9% had an Eastern Cooperative Oncology Group performance status ≥2. Six patients stopped treatment because of toxicity. Grade ≥3 toxicities were: asthenia (16.3%); neutropenia (11.6%); thrombocytopenia (9.3%); diarrhoea (7%), anaemia (4.7%), febrile neutropenia (4.7%) and haematuria (2.3%). In all, 52.4% achieved a ≥30% PSA response and 40.5% had a ≥50% PSA response. The median OS was 15.2 months. CONCLUSION: This prospective pilot study suggests that cabazitaxel 16 mg/m² given 2-weekly has a manageable toxicity profile in docetaxel- and abiraterone acetate-pretreated patients with mCRPC. A prospective phase III trial comparing this regimen with the standard cabazitaxel regimen is planned to confirm these results.

Systematic bacterialization of yeast genes identifies a near-universally swappable pathway.

Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many present-day genes in these lineages predate this divergence, the extent to which these genes still perform their ancestral functions is largely unknown. To test principles governing retention of ancient function, we asked if prokaryotic genes could replace their essential eukaryotic orthologs. We systematically replaced essential genes in yeast by their 1:1 orthologs from Escherichia coli. After accounting for mitochondrial localization and alternative start codons, 31 out of 51 bacterial genes tested (61%) could complement a lethal growth defect and replace their yeast orthologs with minimal effects on growth rate. Replaceability was determined on a pathway-by-pathway basis; codon usage, abundance, and sequence similarity contributed predictive power. The heme biosynthesis pathway was particularly amenable to inter-kingdom exchange, with each yeast enzyme replaceable by its bacterial, human, or plant ortholog, suggesting it as a near-universally swappable pathway.

Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.

Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing. In this study, oxygen microsensors to measure oxygen transects through in vitro cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse wound model, and ex vivo human chronic wound specimens was used. The results showed that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17 to 72 mmHg on live mice and from 6.4 to 1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. To characterize the metabolic activities of the bacteria in the mouse scabs, transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds was performed. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results also indicated that the bacteria within the wounds experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results supported the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions, through their metabolic activities and through their recruitment of cells that consume oxygen for host defensive processes.

Endoscopic endonasal greater palatine artery cauterization at the incisive foramen for control of anterior epistaxis.

OBJECTIVES/HYPOTHESIS: To describe the anatomy of the incisive foramen and the transnasal endoscopic approach to the greater palatine artery at this foramen, and to evaluate the importance of the greater palatine artery as a cause of recurrent anterior epistaxis. STUDY DESIGN: Anatomical dissection, radiographic study, and prospective case series. SETTING: Academic Medical Center. METHODS: Sixty-nine computed tomography scans were reviewed, and measurements were made of the incisive foramina's distance to the anterior nasal spine and subnasale. Twenty-two cadavers had sagittal split craniotomies performed prior to the measurements. The distance from the anterior nasal spine to the incisive foramen was documented. We also present an illustrative case series of patients who underwent endoscopic cautery of the greater palatine artery at the incisive foramen. RESULTS: Radiographic review of the incisive foramen revealed a mean anterior nasal spine to incisive foramen distance on the right and left of 7.9 and 8.1 mm, respectively. The mean distance from the subnasale to incisive foramen on the right and left were 24.7 and 24.9 mm, respectively. CONCLUSIONS: Endoscopic cauterization of the greater palatine artery at the incisive foramen is a safe and effective method to control recurrent anterior epistaxis. The incisive foramen can be predictively found within 1 cm of the anterior nasal spine. Our case series corroborates the above. LEVEL OF EVIDENCE: 4. Laryngoscope, 126:1033-1038, 2016.

The Efficacy of Mitomycin C in the Treatment of Laryngotracheal Stenosis: Results and Experiences with a Difficult Disease Entity.

BACKGROUND/AIMS: Laryngotracheal stenosis (LTS) is a difficult entity to treat, with many patients requiring multiple procedures. Our study assessed the efficacy of mitomycin C (MMC) application as complimentary treatment of LTS. METHODS: We reviewed clinical charts of patients with operative procedures for LTS between January 2005 and May 2013. Patients were grouped according to mitomycin use. Several outcome measures were assessed, including the number of procedures and time between procedures. RESULTS: Seventy-one patients were included in the study (30 MMC, 41 non-MMC). They underwent similar numbers of procedures (2.3 MMC, 2.0 non-MMC, p > 0.05). The average time between procedures was 360 (MMC) and 178 (non-MMC) days (p = 0.015). Multiple treatments with mitomycin increased the duration between procedures (366 vs. 340 days, multiple vs. single application, p > 0.05). Fewer mitomycin patients underwent procedures for respiratory distress than non-MMC patients (6.6 vs. 19.5%, p > 0.05). Mitomycin use increased the duration between procedures in patients treated specifically for subglottic stenosis (375 vs. 186 days, p > 0.05). CONCLUSION: Our results and experiences with mitomycin demonstrate it is an effective agent for adjuvant treatment of LTS. Its use lengthens the symptom-free period, with further improvement demonstrated when patients expected to undergo multiple surgeries receive multiple mitomycin treatments.

The effects of wild blueberry consumption on plasma markers and gene expression related to glucose metabolism in the obese Zucker rat.

Impaired fasting blood glucose is one of the landmark signs of metabolic syndrome, together with hyperinsulinemia, dyslipidemia, hypertension, and a chronic proinflammatory, pro-oxidative, and prothrombotic environment. This study investigates the effect of wild blueberry (WB) consumption on blood glucose levels and other parameters involved in glucose metabolism in the obese Zucker rat (OZR), an experimental model of metabolic syndrome. Sixteen OZRs and 16 lean littermate controls (lean Zucker rat [LZR]) were fed an 8% enriched WB diet or a control (C) diet for 8 weeks. Plasma concentrations of glucose, insulin, glycated hemoglobin GHbA1c, resistin, and retinol-binding protein 4 (RBP4) were measured. Expression of the resistin, RBP4, and glucose transporter GLUT4 genes was also determined both in the liver and the abdominal adipose tissue (AAT). Plasma glycated hemoglobin HbA1c, RBP4, and resistin concentrations were significantly lower in OZRs following the WB diet (-20%, -22%, and -27%, respectively, compared to C diet, P<.05). Following WB consumption, resistin expression was significantly downregulated in the liver of both OZRs and LZRs (-28% and -61%, respectively, P<.05), while RBP4 expression was significantly downregulated in the AAT of both OZRs and LZRs (-87% and -43%, respectively, P<.05). All other markers were not significantly affected following WB consumption. In conclusion, WB consumption normalizes some markers related to glucose metabolism in the OZR model of metabolic syndrome, but has no effect on fasting blood glucose or insulin concentrations.

Impact of 13-valent pneumococcal conjugate vaccine on otitis media bacteriology.

OBJECTIVES: To determine the microbiology of otitis media (OM) since the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in February 2010. METHODS: Middle ear effusion from a pediatric Otolaryngology population undergoing pressure equalization tube (PET) placement was obtained and sent for aerobic culture and antibiotic susceptibility testing between August 2012 and April 2013. Vaccination records were obtained and statistical analysis was completed. RESULTS: During the 8-month period, 236 ears were evaluated, and of those 39 ears were found to have positive cultures. The single nonvaccine Streptococcus pneumoniae (serotype 16) isolate was obtained from a PCV7-only vaccinated patient and was penicillin susceptible. The three most common isolates were Staphylococcus coagulase negative (57%), Haemophilus influenzae (17%), and Moraxella catarrhalis (7%). CONCLUSIONS: This study is the first to assess the bacteriology of OM in a pediatric population undergoing PET placement in the immediate post-PCV13 era. Our study is limited by sample size; however, the lack of S. pneumoniae cultures indicates that PCV13 has had a significant impact on pneumococcal infections during these initial years following licensure.

Revisiting and revising the purinosome.

Some metabolic pathway enzymes are known to organize into multi-enzyme complexes for reasons of catalytic efficiency, metabolite channeling, and other advantages of compartmentalization. It has long been an appealing prospect that de novo purine biosynthesis enzymes form such a complex, termed the "purinosome." Early work characterizing these enzymes garnered scarce but encouraging evidence for its existence. Recent investigations led to the discovery in human cell lines of purinosome bodies-cytoplasmic puncta containing transfected purine biosynthesis enzymes, which were argued to correspond to purinosomes. New discoveries challenge both the functional and physiological relevance of these bodies in favor of protein aggregation.

Temporal maps in appetitive Pavlovian conditioning.

Previous research suggests animals may integrate temporal information into mental representations, or temporal maps. We examined the parameters under which animals integrate temporal information in three appetitive conditioning experiments. In Experiment 1 the temporal relationship between 2 auditory cues was established during sensory preconditioning (SPC). Subsequently, rats were given first order conditioning (FOC) with one of the cues. Results showed integration of the order of cues between the SPC and FOC training phases. In subsequent experiments we tested the hypothesis that quantitative temporal information can be integrated across phases. In Experiment 2, SPC of two short auditory cues superimposed on a longer auditory cue was followed by FOC of either one of the short cues, or of the long cue at different times in the cue. Contrary to our predictions we did not find evidence of integration of temporal information across the phases of the experiment and instead responding to the SPC cues in Experiment 2 appeared to be dominated by generalization from the FOC cues. In Experiment 3 shorter auditory cues were superimposed on a longer duration light cue but with asynchronous onset and offset of the superimposed cues. There is some evidence consistent with the hypothesis that quantitative discrimination of whether reward should be expected during the early or later parts of a cue could be integrated across experiences. However, the pattern of responding within cues was not indicative of integration of quantitative temporal information. Generalization of expected times of reward during FOC seems to be the dominant determinant of within-cue response patterns in these experiments. Consequently, while we clearly demonstrated the integration of temporal order in the modulation of this dominant pattern we did not find strong evidence of integration of precise quantitative temporal information. This article is part of a Special Issue entitled: Associative and Temporal Learning.