Revealing the Activity of Trimeric G-proteins in Live Cells with a Versatile Biosensor Design.

Heterotrimeric G-proteins (Gαßγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gßγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.

Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells.

Chimeric antigen receptors (CARs) redirect T cell cytotoxicity against cancer cells, providing a promising approach to cancer immunotherapy. Despite extensive clinical use, the attributes of CAR co-stimulatory domains that impact persistence and resistance to exhaustion of CAR-T cells remain largely undefined. Here, we report the influence of signaling domains of coreceptors CD28 and 4-1BB on the metabolic characteristics of human CAR T cells. Inclusion of 4-1BB in the CAR architecture promoted the outgrowth of CD8(+) central memory T cells that had significantly enhanced respiratory capacity, increased fatty acid oxidation and enhanced mitochondrial biogenesis. In contrast, CAR T cells with CD28 domains yielded effector memory cells with a genetic signature consistent with enhanced glycolysis. These results provide, at least in part, a mechanistic insight into the differential persistence of CAR-T cells expressing 4-1BB or CD28 signaling domains in clinical trials and inform the design of future CAR T cell therapies.

Improved Speech Hearing in Noise with Invasive Electrical Brain Stimulation.

Speech perception in noise is a challenging everyday task with which many listeners have difficulty. Here, we report a case in which electrical brain stimulation of implanted intracranial electrodes in the left planum temporale (PT) of a neurosurgical patient significantly and reliably improved subjective quality (up to 50%) and objective intelligibility (up to 97%) of speech in noise perception. Stimulation resulted in a selective enhancement of speech sounds compared with the background noises. The receptive fields of the PT sites whose stimulation improved speech perception were tuned to spectrally broad and rapidly changing sounds. Corticocortical evoked potential analysis revealed that the PT sites were located between the sites in Heschl's gyrus and the superior temporal gyrus. Moreover, the discriminability of speech from nonspeech sounds increased in population neural responses from Heschl's gyrus to the PT to the superior temporal gyrus sites. These findings causally implicate the PT in background noise suppression and may point to a novel potential neuroprosthetic solution to assist in the challenging task of speech perception in noise.SIGNIFICANCE STATEMENT Speech perception in noise remains a challenging task for many individuals. Here, we present a case in which the electrical brain stimulation of intracranially implanted electrodes in the planum temporale of a neurosurgical patient significantly improved both the subjective quality (up to 50%) and objective intelligibility (up to 97%) of speech perception in noise. Stimulation resulted in a selective enhancement of speech sounds compared with the background noises. Our local and network-level functional analyses placed the planum temporale sites in between the sites in the primary auditory areas in Heschl's gyrus and nonprimary auditory areas in the superior temporal gyrus. These findings causally implicate planum temporale in acoustic scene analysis and suggest potential neuroprosthetic applications to assist hearing in noise.

Sepsis Program Activities in Acute Care Hospitals - National Healthcare Safety Network, United States, 2022.

Sepsis, life-threatening organ dysfunction secondary to infection, contributes to at least 1.7 million adult hospitalizations and at least 350,000 deaths annually in the United States. Sepsis care is complex, requiring the coordination of multiple hospital departments and disciplines. Sepsis programs can coordinate these efforts to optimize patient outcomes. The 2022 National Healthcare Safety Network (NHSN) annual survey evaluated the prevalence and characteristics of sepsis programs in acute care hospitals. Among 5,221 hospitals, 3,787 (73%) reported having a committee that monitors and reviews sepsis care. Prevalence of these committees varied by hospital size, ranging from 53% among hospitals with 0-25 beds to 95% among hospitals with >500 beds. Fifty-five percent of all hospitals provided dedicated time (including assigned protected time or job description requirements) for leaders of these committees to manage a program and conduct daily activities, and 55% of committees reported involvement with antibiotic stewardship programs. These data highlight opportunities, particularly in smaller hospitals, to improve the care and outcomes of patients with sepsis in the United States by ensuring that all hospitals have sepsis programs with protected time for program leaders, engagement of medical specialists, and integration with antimicrobial stewardship programs. CDC's Hospital Sepsis Program Core Elements provides a guide to assist hospitals in developing and implementing effective sepsis programs that complement and facilitate the implementation of existing clinical guidelines and improve patient care. Future NHSN annual surveys will monitor uptake of these sepsis core elements.

Chemical neurolysis of the genicular nerves for chronic refractory knee pain: an observational cohort study.

OBJECTIVE: Evaluate outcomes of genicular nerve chemical neurolysis (GChN) in a real-world population with chronic knee pain. DESIGN: Restrospective, observational cohort study. SETTING: Tertiary academic medical center. SUBJECTS: Consecutive patients who had undergone GChN ≥3 months prior. METHODS: Standardized surveys were collected by telephone and included the numerical rating scale, opioid analgesic use, and Patient Global Impression of Change. Age, sex, body mass index, duration of pain, history of arthroplasty, lack of effect from previous radiofrequency ablation, percentage relief from a prognostic block, and volume of phenol used at each injection site were extracted from charts. Descriptive statistics were calculated, and logistic regression analyses were performed to identify factors influencing treatment outcome. RESULTS: At the time of follow-up after GChN (mean ± SD: 9.9 ± 6.1 months), 43.5% (95% CI = 33.5-54.1) of participants reported ≥50% sustained pain reduction. On the Patient Global Impression of Change assessment, 45.9% (95% CI = 35.5-56.7) of participants reported themselves to be "very much improved" or "much improved." Of 40 participants taking opioids at baseline, 11 (27.5%; 95% CI = 14.6-43.9) ceased use. Of participants with a native knee treated, 46.3% reported ≥50% pain reduction, whereas of participants with an arthroplasty in the treated knee, 33.3% reported this threshold of pain reduction (P = .326). Logistic regression analyses did not reveal associations between treatment success and any of the factors that we evaluated. CONCLUSIONS: GChN could provide a robust and durable treatment effect in a subset of individuals with chronic knee pain with complicating factors traditionally associated with poor treatment outcomes, such as those with pain refractory to radiofrequency ablation or those who have undergone arthroplasty.

Deep-Sea Sponges and Corals off the Western Coast of Florida-Intracellular Mechanisms of Action of Bioactive Compounds and Technological Advances Supporting the Drug Discovery Pipeline.

The majority of natural products utilized to treat a diverse array of human conditions and diseases are derived from terrestrial sources. In recent years, marine ecosystems have proven to be a valuable resource of diverse natural products that are generated to defend and support their growth. Such marine sources offer a large opportunity for the identification of novel compounds that may guide the future development of new drugs and therapies. Using the National Oceanic and Atmospheric Administration (NOAA) portal, we explore deep-sea coral and sponge species inhabiting a segment of the U.S. Exclusive Economic Zone, specifically off the western coast of Florida. This area spans ~100,000 km2, containing coral and sponge species at sea depths up to 3000 m. Utilizing PubMed, we uncovered current knowledge on and gaps across a subset of these sessile organisms with regards to their natural products and mechanisms of altering cytoskeleton, protein trafficking, and signaling pathways. Since the exploitation of such marine organisms could disrupt the marine ecosystem leading to supply issues that would limit the quantities of bioactive compounds, we surveyed methods and technological advances that are necessary for sustaining the drug discovery pipeline including in vitro aquaculture systems and preserving our natural ecological community in the future. Collectively, our efforts establish the foundation for supporting future research on the identification of marine-based natural products and their mechanism of action to develop novel drugs and therapies for improving treatment regimens of human conditions and diseases.

Black fungus outbreak in India - A direct consequence of COVID-19 surge: A myth or reality.

The deadly second wave of COVID-19 has seen an unprecedented surge in mucormycosis associated mortality in India, overwhelming the heath authorities with challenges beyond measure. Also known as black fungus, this life-threatening fungal infection usually manifests in the nose, spreads to the eyes, and in some cases also to the brain. Immune suppression, pre-existing conditions, prolonged and indiscriminate use of steroids, and unhygienic environments are some of the widely recognized risk factors for contracting black fungus in individuals recovered from COVID-19. However, diagnosis of the infection remains insufficient due to the lack a holistic understanding of the possible risks, symptoms, and exposure pathways and therefore no definite protocol exists for managing this fatal infection. Here, we synthesize the current state of knowledge on black fungus outbreak in India and identify key gaps in its understanding with respect to potential risk factors leading to the widespread infection. We looked at 3354 black fungus cases in India, enlisting ailment history (particularly diabetes) and steroid usage in COVID-19 patients as the key factors responsible for exacerbating risks associated with the disease. However, we also press on the possibilities that other less studied non-traditional risk factors may also have a role in causing the infection. Black fungus is therefore a reality of COVID-19, with or without diabetes or steroid use needs to be investigated. We believe such a review is imperative for making informed decisions specially around timely diagnosis and channelizing efforts in controlling the spread of COVID-19 associated mucormycosis.

Oral cancer patients achieve comparable survival at high safety-net burden hospitals.

PURPOSE: To evaluate the impact of hospital safety-net burden and social demographics on the overall survival of patients with oral cavity squamous cell carcinoma. MATERIALS AND METHODS: We identified 48,176 oral cancer patients diagnosed between the years 2004 to 2015 from the National Cancer Database and categorized treatment facilities as no, low, or high safety-net burden hospitals based on the percentage of uninsured or Medicaid patients treated. Using the Kaplan Meier method and multivariate analysis, we examined the effect of hospital safety-net burden, sociodemographic variables, and clinical factors on overall survival. RESULTS: Of the 1269 treatment facilities assessed, the median percentage of uninsured/Medicaid patients treated was 0% at no, 11.6% at low, and 23.5% at high safety-net burden hospitals and median survival was 68.6, 74.8, and 55.0 months, respectively (p < 0.0001). High safety-net burden hospitals treated more non-white populations (15.4%), lower median household income (<$30,000) (23.2%), and advanced stage cancers (AJCC III/IV) (54.6%). Patients treated at low (aHR = 0.97; 95% CI = 0.91-1.04, p = 0.405) and high (aHR = 1.05; 95% CI = 0.98-1.13, p = 0.175) safety-net burden hospitals did not experience worse survival outcomes compared to patients treated at no safety-net burden hospitals. CONCLUSION: High safety-net burden hospitals treated more oral cancer patients of lower socioeconomic status and advanced disease. Multivariate analysis showed high safety-net burden hospitals achieved comparable patient survival to lower burden hospitals.

Functional characterization of human Heschl's gyrus in response to natural speech.

Heschl's gyrus (HG) is a brain area that includes the primary auditory cortex in humans. Due to the limitations in obtaining direct neural measurements from this region during naturalistic speech listening, the functional organization and the role of HG in speech perception remain uncertain. Here, we used intracranial EEG to directly record neural activity in HG in eight neurosurgical patients as they listened to continuous speech stories. We studied the spatial distribution of acoustic tuning and the organization of linguistic feature encoding. We found a main gradient of change from posteromedial to anterolateral parts of HG. We also observed a decrease in frequency and temporal modulation tuning and an increase in phonemic representation, speaker normalization, speech sensitivity, and response latency. We did not observe a difference between the two brain hemispheres. These findings reveal a functional role for HG in processing and transforming simple to complex acoustic features and inform neurophysiological models of speech processing in the human auditory cortex.

Delayed onset of tooth decay in a routine pediatric adenotonsillectomy.

Adenotonsillectomy is a common pediatric surgical procedure with a well-defined safety profile. Major complications from this procedure include bleeding/hemorrhage, infection, pain leading to dehydration, and airway obstruction or edema. Though rare, oral endotracheal intubation and oral retractor placement may result in injuries to the teeth and the surrounding soft tissue. We describe a rare case of delayed tooth decay in a 3-year-old female following an otherwise routine adenotonsillectomy.

Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein.

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gßγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

Atypical activation of the G protein Gαq by the oncogenic mutation Q209P.

The causative role of G protein-coupled receptor (GPCR) pathway mutations in uveal melanoma (UM) has been well-established. Nearly all UMs bear an activating mutation in a GPCR pathway mediated by G proteins of the Gq/11 family, driving tumor initiation and possibly metastatic progression. Thus, targeting this pathway holds therapeutic promise for managing UM. However, direct targeting of oncogenic Gαq/11 mutants, present in ∼90% of UMs, is complicated by the belief that these mutants structurally resemble active Gαq/11 WT. This notion is solidly founded on previous studies characterizing Gα mutants in which a conserved catalytic glutamine (Gln-209 in Gαq) is replaced by leucine, which leads to GTPase function deficiency and constitutive activation. Whereas Q209L accounts for approximately half of GNAQ mutations in UM, Q209P is as frequent as Q209L and also promotes oncogenesis, but has not been characterized at the molecular level. Here, we characterized the biochemical and signaling properties of Gαq Q209P and found that it is also GTPase-deficient and activates downstream signaling as efficiently as Gαq Q209L. However, Gαq Q209P had distinct molecular and functional features, including in the switch II region of Gαq Q209P, which adopted a conformation different from that of Gαq Q209L or active WT Gαq, resulting in altered binding to effectors, Gßγ, and regulators of G-protein signaling (RGS) proteins. Our findings reveal that the molecular properties of Gαq Q209P are fundamentally different from those in other active Gαq proteins and could be leveraged as a specific vulnerability for the ∼20% of UMs bearing this mutation.

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.

The neutrophil to albumin ratio as a predictor of pathological complete response in rectal cancer patients following neoadjuvant chemoradiation.

Pathological complete response (pCR) following neoadjuvant chemoradiotherapy (nCRT) and total mesorectal excision (TME), in patients with locally advanced rectal cancer, occurs in 15-27% of patients. Because blood cell counts and albumin are a direct indicator of the host environment, a response to nCRT might be predicted by these markers. This study was carried out to determine whether the neutrophil to albumin ratio (NAR) was predictive of pCR in veteran patients. Ninety-eight patients with rectal cancer who underwent standard nCRT, followed by TME were analyzed. Pre-nCRT and post-nCRT hematologic data were collected. Univariate and multivariate analyses were carried out. Kaplan-Meier curves were constructed with our primary endpoint of pCR. Male patients (99%), age 62.4±9.1 years, BMI=27.4±5.9 kg/m, rectal cancer distance from anal verge=7.1±4.5 cm (SD), interval between nCRT and TME=8 weeks, 55% patients=low anterior resection, 95% received 5-fluorouracil, and all patients received radiation, with 15% achieving a pCR. Univariate analysis showed that pre-nCRT carcinoembryonic antigen (15.8±45.1 vs. 3.5±5.3 ng/dl; P=0.002) and the pre-nCRT NAR (16.4±4.8 vs. 14.2±1.6; P=0.002) were associated with pCR. On multivariate analysis, pre-nCRT carcinoembryonic antigen (odds ratio=0.41, 95% confidence interval 0.22-0.77) and pre-nCRT NAR (odds ratio=0.76, 95% confidence interval 0.60-0.97) remained independent predictors of pCR. Overall survival between nonresponders and pCR patients at 1, 5, and 10 years was 96, 62, and 44% versus 93, 85, and 61%, P=0.13, and disease-free survival was 95, 60, and 47% versus 93, 85, and 61%, P=0.17; respectively. Our study shows that the pre-nCRT NAR is an independent predictor of pCR. These findings should be applied to other cohorts to determine its validity and reliability for use as a potential predictor of pCR.

Extended release of ketotifen from silica shell nanoparticle-laden hydrogel contact lenses: in vitro and in vivo evaluation.

Ketotifen an anti-allergic drug delivered via eye drops has major limitations, including poor ocular bioavailability and poor patient compliance. The objective of the research work was to fabricate ketotifen loaded microemulsion laden hydrogels and silica shell nanoparticle-laden (prepared from microemulsion using octyltrimethoxysilane) hydrogels to achieve extended ocular drug delivery. The porous silica shell membrane was synthesized at the liquid interface of microemulsion, which facilitates the prolongation of drug release duration from hydrogels. Drug encapsulated microemulsion and silica shell nanoparticles were dispersed separately in pre-monomer mixture, and fabricated to hydrogel. For comparison, hydrogel with direct drug entrapment was also fabricated. Significant loss in transmittance and physical properties was observed in hydrogels with direct drug entrapment. While, microemulsion and silica shell nanoparticle-laden hydrogels did not show significant effect on transmittance and physical properties. The in vitro drug release data showed extended release of ketotifen from hydrogels in following order: direct loading