Ancestry matters: Building inclusivity into preclinical study design.

Human cell lines (CLs) are key assets for biomedicine but lack ancestral diversity. Here, we explore why genetic diversity among cell-based models is essential for making preclinical research more equitable and widely translatable. We lay out practical actions that can be taken to improve inclusivity in study design.

Cell line authentication: a necessity for reproducible biomedical research.

Immortalized or continuous cell lines are invaluable tools in basic and preclinical research. However, the widespread use of misidentified cell lines is a serious threat to scientific reproducibility. Based on the experiences of mandatory cell line authentication at the International Journal of Cancer (IJC), we provide an overview of the issues pertinent to misidentified cell lines and discuss available solutions. We also summarize the lessons learned, revealing that at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified. About 4% of the considered manuscripts are rejected for severe cell line problems, and most are subsequently published in other journals. In order to diminish such malpractice and its consequences for the scientific record, we postulate that strict multi-layered quality control is essential. Besides journals and publishers, we encourage scientists, research institutions, and funders to take action on the matter and revise their respective policies. Hence, we provide concrete recommendations on introducing regular authentication schemes and staff training, and discuss future steps for enhancing good cell culture practices.

Neutralization profiles of HIV-1 viruses from the VRC01 Antibody Mediated Prevention (AMP) trials.

The VRC01 Antibody Mediated Prevention (AMP) efficacy trials conducted between 2016 and 2020 showed for the first time that passively administered broadly neutralizing antibodies (bnAbs) could prevent HIV-1 acquisition against bnAb-sensitive viruses. HIV-1 viruses isolated from AMP participants who acquired infection during the study in the sub-Saharan African (HVTN 703/HPTN 081) and the Americas/European (HVTN 704/HPTN 085) trials represent a panel of currently circulating strains of HIV-1 and offer a unique opportunity to investigate the sensitivity of the virus to broadly neutralizing antibodies (bnAbs) being considered for clinical development. Pseudoviruses were constructed using envelope sequences from 218 individuals. The majority of viruses identified were clade B and C; with clades A, D, F and G and recombinants AC and BF detected at lower frequencies. We tested eight bnAbs in clinical development (VRC01, VRC07-523LS, 3BNC117, CAP256.25, PGDM1400, PGT121, 10-1074 and 10E8v4) for neutralization against all AMP placebo viruses (n = 76). Compared to older clade C viruses (1998-2010), the HVTN703/HPTN081 clade C viruses showed increased resistance to VRC07-523LS and CAP256.25. At a concentration of 1µg/ml (IC80), predictive modeling identified the triple combination of V3/V2-glycan/CD4bs-targeting bnAbs (10-1074/PGDM1400/VRC07-523LS) as the best against clade C viruses and a combination of MPER/V3/CD4bs-targeting bnAbs (10E8v4/10-1074/VRC07-523LS) as the best against clade B viruses, due to low coverage of V2-glycan directed bnAbs against clade B viruses. Overall, the AMP placebo viruses represent a valuable resource for defining the sensitivity of contemporaneous circulating viral strains to bnAbs and highlight the need to update reference panels regularly. Our data also suggests that combining bnAbs in passive immunization trials would improve coverage of global viruses.

COVID-19 vaccine efficacy in participants with weakened immune systems from four randomized-controlled trials.

BACKGROUND: Although the SARS-CoV-2 vaccines are highly efficacious at preventing severe disease in the general population, current data are lacking regarding vaccine efficacy (VE) for individuals with mild immunocompromising conditions. METHODS: A post-hoc, cross-protocol analysis of participant-level data from the blinded phase of four randomized, placebo-controlled, COVID-19 vaccine phase 3 trials (Moderna, AstraZeneca, Janssen, and Novavax) was performed. We defined a "tempered immune system" (TIS) variable via a consensus panel based on medical history and medications to determine VE against symptomatic and severe COVID-19 cases in TIS participants versus non-TIS (NTIS) individuals starting at 14 days after completion of the primary series through the blinded phase for each of the four trials. An analysis of participants living with well-controlled HIV was conducted using the same methods. RESULTS: 3,852/30,351 (12.7%) Moderna participants, 3,088/29,868 (10.3%) Novavax participants, 3,549/32,380 (11.0%) AstraZeneca participants, and 5,047/43,788 (11.5%) Janssen participants were identified as having a TIS. Most TIS conditions (73.9%) were due to metabolism and nutritional disorders. Vaccination (versus placebo) significantly reduced the likelihood of symptomatic and severe COVID-19 for all participants for each trial. VE was not significantly different for TIS participants vs NTIS for either symptomatic or severe COVID-19 for each trial, nor was VE significantly different in the symptomatic endpoint for participants with HIV. CONCLUSIONS: For individuals with mildly immunocompromising conditions, there is no evidence of differences in VE against symptomatic or severe COVID-19 compared to those with non-tempered immune systems in the four COVID-19 vaccine randomized controlled efficacy trials.

Misspellings or "miscellings"-Non-verifiable and unknown cell lines in cancer research publications.

Reproducible laboratory research relies on correctly identified reagents. We have previously described gene research papers with wrongly identified nucleotide sequence(s), including papers studying miR-145. Manually verifying reagent identities in 36 recent miR-145 papers found that 56% and 17% of papers described misidentified nucleotide sequences and cell lines, respectively. We also found 5 cell line identifiers in miR-145 papers with misidentified nucleotide sequences and cell lines, and 18 cell line identifiers published elsewhere, that did not represent indexed human cell lines. These 23 identifiers were described as non-verifiable (NV), as their identities were unclear. Studying 420 papers that mentioned 8 NV identifier(s) found 235 papers (56%) that referred to 7 identifiers (BGC-803, BSG-803, BSG-823, GSE-1, HGC-7901, HGC-803, and MGC-823) as independent cell lines. We could not find any publications describing how these cell lines were established. Six cell lines were sourced from cell line repositories with externally accessible online catalogs, but these cell lines were not indexed as claimed. Some papers also stated that short tandem repeat (STR) profiles had been generated for three cell lines, yet no STR profiles could be identified. In summary, as NV cell lines represent new challenges to research integrity and reproducibility, further investigations are required to clarify their status and identities.

Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia.

Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3' region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.

Reversible Hydrosulfide (HS-) Binding Using Exclusively C-H Hydrogen-Bonding Interactions in Imidazolium Hosts.

H2S is a physiologically important signaling molecule with complex roles in biology and exists primarily as HS- at physiological pH. Despite this anionic character, few investigations have focused on the molecular recognition and reversible binding of this important biological anion. Using a series of imidazole and imidazolium host molecules, we investigate the role of preorganization and charge on HS- binding. Using a macrocyclic bis-imidazolium receptor, we demonstrate the unexpected 2:1 host-guest binding of HS-, which was characterized both in solution and by X-ray crystallography. To the best of our knowledge, this is the first example of this binding stoichiometry for HS- binding. Moreover, the short C-H···S distances of 2.53, 2.54, 2.76, and 2.79 Å are well within the sum of the van der Waals radii of the interacting atoms, which is consistent with strong C-H···S interactions.

Protein dose-sparing effect of AS01B adjuvant in a randomized preventive HIV vaccine trial of ALVAC-HIV (vCP2438) and adjuvanted bivalent subtype C gp120.

BACKGROUND: HVTN 120 is a phase 1/2a randomized double-blind placebo-controlled HIV vaccine trial that evaluated the safety and immunogenicity of ALVAC-HIV (vCP2438) and MF59- or AS01B-adjuvanted bivalent subtype C gp120 Env protein at two dose levels in healthy HIV-uninfected adults. Trial registration URL https://clinicaltrials.gov/ct2/show/NCT03122223 and registration number NCT03122223. METHODS: Participants received ALVAC-HIV (vCP2438) alone or placebo at months 0 and 1. At months 3 and 6, participants received either placebo, ALVAC-HIV (vCP2438) with 200µg of bivalent subtype C gp120 adjuvanted with MF59 or AS01B, or ALVAC-HIV (vCP2438) with 40µg of bivalent subtype C gp120 adjuvanted with AS01B. Primary outcomes were safety and immune responses. RESULTS: We enrolled 160 participants, 55% females, 18-40 years old (median age 24 years) of whom 150 received vaccine and 10 placebo. Vaccines were generally safe and well tolerated. At months 6.5 and 12, CD4+ T-cell response rates and magnitudes were higher in the AS01B-adjuvanted groups than in the MF59-adjuvanted group. At month 12, HIV-specific Env-gp120 binding antibody response magnitudes in the 40µg gp120/AS01B group were higher than in either of the 200µg gp120 groups. CONCLUSIONS: The 40µg dose gp120/AS01B regimen elicited the highest CD4+ T-cell and binding antibody responses.

Intramolecular Diels-Alder Reaction of a Biphenyl Group in a Strained meta-Quaterphenylene Acetylene.

At elevated temperatures, a strained, cyclic meta-quaterphenylene acetylene undergoes an intramolecular cyclization reaction to form benz[e]indeno[1,2,3-hi]acephenanthrylene. This reaction represents an example of a Diels-Alder reaction at the 2-, 1-, 1'-, and 2'-positions of a biphenyl derivative, a region analogous to the bay regions of perylene and other periacenes. The reaction proceeds cleanly with high conversion. Kinetics studies of a methylated derivative reveal that the ΔG‡ for the reaction is ∼40-41 kcal/mol, and computational models predict a similar value of Grel for the transition state of a concerted [4 + 2]-cycloaddition.

Cervicovaginal Tissue Residence Confers a Distinct Differentiation Program upon Memory CD8 T Cells.

Tissue-resident memory CD8 T cells (CD8 TRM) are critical for maintaining barrier immunity. CD8 TRM have been mainly studied in the skin, lung and gut, with recent studies suggesting that the signals that control tissue residence and phenotype are highly tissue dependent. We examined the T cell compartment in healthy human cervicovaginal tissue (CVT) and found that most CD8 T cells were granzyme B+ and TCF-1- To address if this phenotype is driven by CVT tissue residence, we used a mouse model to control for environmental factors. Using localized and systemic infection models, we found that CD8 TRM in the mouse CVT gradually acquired a granzyme B+, TCF-1- phenotype as seen in human CVT. In contrast to CD8 TRM in the gut, these CD8 TRM were not stably maintained regardless of the initial infection route, which led to reductions in local immunity. Our data show that residence in the CVT is sufficient to progressively shape the size and function of its CD8 TRM compartment.

Incidence and outcomes of critical illness in indigenous peoples: a systematic review and meta-analysis.

BACKGROUND: Indigenous Peoples experience health inequities and racism across the continuum of health services. We performed a systematic review and meta-analysis of the incidence and outcomes of critical illness among Indigenous Peoples. METHODS: We searched Ovid MEDLINE/PubMed, Ovid EMBASE, Google Scholar, and Cochrane Central Register of Controlled Trials (inception to October 2022). Observational studies, case series of > 100 patients, clinical trial arms, and grey literature reports of Indigenous adults were eligible. We assessed risk of bias using the Newcastle-Ottawa Scale and appraised research quality from an Indigenous perspective using the Aboriginal and Torres Strait Islander Quality Assessment Tool. ICU mortality, ICU length of stay, and invasive mechanical ventilation (IMV) were compared using risk ratios and mean difference (MD) for dichotomous and continuous outcomes, respectively. ICU admission was synthesized descriptively. RESULTS: Fifteen studies (Australia and/or New Zealand [n = 12] and Canada [n = 3]) were included. Risk of bias was low in 10 studies and moderate in 5, and included studies had minimal incorporation of Indigenous perspectives or consultation. There was no difference in ICU mortality between Indigenous and non-Indigenous (RR 1.14, 95%CI 0.98 to 1.34, I2 = 87%). We observed a shorter ICU length of stay among Indigenous (MD - 0.25; 95%CI, - 0.49 to - 0.00; I2 = 95%) and a higher use for IMV among non-Indigenous (RR 1.10; 95%CI, 1.06 to 1.15; I2 = 81%). CONCLUSION: Research on Indigenous Peoples experience with critical care is poorly characterized and has rarely included Indigenous perspectives. ICU mortality between Indigenous and non-Indigenous populations was similar, while there was a shorter ICU length of stay and less mechanical ventilation use among Indigenous patients. Systematic Review Registration PROSPERO CRD42021254661; Registered: 12 June, 2021.

Finding social need-les in a haystack: ascertaining social needs of Medicare patients recorded in the notes of care managers.

BACKGROUND: Unmet social needs may impair health and access to health care, and intervening on these holds particular promise in high-risk patient populations, such as those with multiple chronic conditions. Our objective was to identify social needs in a patient population at significant risk-Medicare enrollees with multiple chronic illnesses enrolled in care management services-and measure their prevalence prior to any systematic screening. METHODS: We partnered with Renova Health, an independent Medicare Chronic Care Management (CCM) provider with patients in 10 states during our study period (January 2017 through August 2020). Our data included over 3,000 Medicare CCM patients, representing nearly 20,000 encounters. We used a dictionary-based natural language processing approach to ascertain the prevalence of six domains of barriers to care (food insecurity, housing instability, utility hardship) and unmet social needs (health care affordability, need for supportive services, transportation) in notes taken during telephonic Medicare CCM patient encounters. RESULTS: Barriers to care, specifically need for supportive services (2.4%) and health care affordability (0.8%), were the most prevalent domains identified. Transportation as a barrier to care came up relatively less frequently in CCM encounters (0.1%). Unmet social needs were identified at a comparatively lower rate, with potential housing instability (0.3%) flagged most followed by potential utility hardship (0.2%) and food insecurity (0.1%). CONCLUSIONS: There is substantial untapped opportunity to systematically screen for social determinants of health and unmet social needs in care management.

Anti-Inflammatory and Antimicrobial Properties of Thyme Oil and Its Main Constituents.

Thyme oil (TO) is derived from the flowers of various plants belonging to the genus Thymus. It has been used as a therapeutic agent since ancient times. Thymus comprises numerous molecular species exhibiting diverse therapeutic properties that are dependent on their biologically active concentrations in the extracted oil. It is therefore not surprising that oils extracted from different thyme plants present different therapeutic properties. Furthermore, the phenophase of the same plant species has been shown to yield different anti-inflammatory properties. Given the proven efficacy of TO and the diversity of its constituents, a better understanding of the interactions of the various components is warranted. The aim of this review is to gather the latest research findings regarding TO and its components with respect to their immunomodulatory properties. An optimization of the various components has the potential to yield more effective thyme formulations with increased potency.

Validation of the Black Community Activism Orientation Scale with racially and ethnically diverse college students.

This study fills a methodological gap in racial justice research by assessing the utility and validity of the Black Community Activism Orientation Scale (BCAOS) in a racially and ethnically diverse sample of college-going young adults (N = 624, M = 19.4 years, SD = 1.89) from 10 colleges in the United States. Confirmatory factor analysis was conducted to estimate the goodness of fit of the proposed three-factor model and assess the validity of the BCAOS. Findings from the confirmatory factor analysis provide statistical support for use of the BCAOS as a measure of racial justice activism in support of Black communities among racially and ethnically diverse college-going young adults. Findings from the study also suggest that White college students and men are less oriented toward racial justice activism than women and racially marginalized students. Convergent and discriminant validity were established through bivariate correlations of the BCAOS factors with other civic development measures. As more and more young people consider the importance of standing against racial oppression, the BCAOS has utility as an assessment instrument in future racial justice research, education, intervention, and youth programming efforts.

Safety and Antiviral Effects of Nebulized PC786 in a Respiratory Syncytial Virus Challenge Study.

BACKGROUND: PC786 is a nebulized nonnucleoside respiratory syncytial virus (RSV) polymerase inhibitor designed to treat RSV, which replicates in the superficial layer of epithelial cells lining the airways. METHODS: Fifty-six healthy volunteers inoculated with RSV-A (Memphis 37b) were randomly dosed with either nebulized PC786 (5 mg) or placebo, twice daily for 5 days, from either 12 hours after confirmation of RSV infection or 6 days after virus inoculation. Viral load (VL), disease severity, pharmacokinetics, and safety were assessed until discharge. RSV infection was confirmed by reverse-transcription quantitative polymerase chain reaction with any positive value (intention-to-treat infected [ITT-I] population) or RSV RNA ≥1 log10 plaque-forming unit equivalents (PFUe)/mL (specific intention-to-treat infection [ITT-IS] population) in nasal wash samples. RESULTS: In the ITT-I population, the mean VL area under the curve (AUC) was lower in the PC786 group than the placebo group (274.1 vs 406.6 log10 PFUe/mL × hour; P = .0359). PC786 showed a trend toward reduction of symptom score and mucous weight. In ITT-IS (post hoc analysis), the latter was statistically significant as well as VL AUC (P = .0126). PC786 showed an early time to maximum plasma concentration, limited systemic exposure, and long half-life and consequently a 2-fold accumulation over the 5-day dosing period. PC786 was well tolerated. CONCLUSIONS: Nebulized PC786 demonstrated a significant antiviral effect against RSV, warranting further clinical study. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov: NCT03382431; EudraCT: 2017-002563-18.

Maturity-Onset Diabetes of the Young: Rapid Evidence Review.

Maturity-onset diabetes of the young (MODY) is a non-insulin-dependent form of diabetes mellitus that is usually diagnosed in young adulthood. MODY is most often an autosomal dominant disease and is divided into subtypes (MODY1 to MODY14) based on the causative genetic mutation. Subtypes 1 to 3 account for 95% of cases. MODY is often misdiagnosed as type 1 or 2 diabetes and should be suspected in nonobese patients who have diabetes that was diagnosed at a young age (younger than 30 years) and a strong family history of diabetes. Unlike those with type 1 diabetes, patients with MODY have preserved pancreatic beta-cell function three to five years after diagnosis, as evidenced by detectable serum C-peptide levels with a serum glucose level greater than 144 mg per dL and no laboratory evidence of pancreatic beta-cell autoimmunity. Patients with MODY1 and MODY3 have progressive hyperglycemia and vascular complication rates similar to patients with types 1 and 2 diabetes. Lifestyle modification including a low-carbohydrate diet should be the first-line treatment for MODY1 and MODY3. Sulfonylureas are the preferred pharmacologic therapy based on pathophysiologic reasoning, although clinical trials are lacking. Patients with MODY2 have mild stable fasting hyperglycemia with low risk of diabetes-related complications and generally do not require treatment, except in pregnancy. Pregnant patients with MODY may require insulin therapy and additional fetal monitoring for macrosomia.

Supporting autonomy in youth mentoring relationships.

The preference for and exercise of autonomous decision-making in adolescence is a normative developmental process. Yet, increased autonomy is associated with both risks and benefits. Connection to others through positive relationships, including mentoring relationships, is one context that predicts healthy autonomous decision-making. In other ways, such relationships can interfere or stifle the development of autonomy. In synthesizing the existing scientific literature on autonomy development and autonomy-supportive practices, we propose a framework for considering the role of mentors in supporting autonomy through five domains of influence: role modeling, encouraging, providing access to resources, relationships, and experiences, advocacy, and conversations about behavior change. We provide suggestions for research and practice.

Exploring civic behaviors amongst college students in a year of national unrest.

This study examined the role of demographics, civic beliefs, and the impact of the COVID-19 pandemic in association with distinct forms of civic participation. College students were recruited across 10 institutions of higher education to complete an online survey. Bivariate, multivariable linear, and logistic regressions were performed. Findings indicated that participants from traditionally marginalized backgrounds were more likely to engage in systemchallenging forms of civic participation and community engagement than those from more privileged backgrounds. Participants who rated high in critical reflection, viewed racism as a key issue, and were heavily impacted by the COVID-19 pandemic were also more likely to engage in system-challenging forms of civic participation. Participants who endorsed beliefs supporting current systems of power were more likely to report they intended to vote. Results highlight implications for antiracist activism, community engagement, and traditional political civic behaviors.

Photoperiod sensing of the circadian clock is controlled by EARLY FLOWERING 3 and GIGANTEA.

ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not only essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mutants of ELF3 and GI have been well studied, their genetic interaction remains unclear. We generated an elf3 gi double mutant to study their genetic relationship in clock-controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth differentially during the night and the day, respectively. Circadian clock assays revealed that ELF3 and GI are essential that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light-dark cycles even under diurnal conditions. Consequently, clock-mediated photoperiod-responsive growth and development are completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod-insensitive crops by adjusting the allelic combination of these two key genes.

Cross contamination meets misclassification: Awakening of CHP-100 from sleeping beauty sleep-A reviewed model for Ewing's sarcoma.

A human cell line of neuroblastic tissue, which was believed to have been lost to science due to its unavailability in public repositories, is revived and reclassified. In the 1970s, a triple set of neuroblastoma (NB) cell lines became available for research as MYCN-amplified vs nonamplified models (CHP-126/-134 and CHP-100, respectively). Confusingly, CHP-100 was used in subsequent years as a model for NB and, since the 1990s, as a model for neuroepithelioma and later as a model for Ewing's sarcoma (ES), which inevitably led to non-reproducible results. A deposit at a bioresource center revealed that globally available stocks of CHP-100 were identical to the prominent NB cell line IMR-32 and CHP-100 was included into the list of misidentified cell lines. Now we report on the rediscovery of an authentic CHP-100 cell line and provide evidence of incorrect classification during establishment. We show that CHP-100 cells carry a t(11;22)(q24;q12) type II EWSR1-FLI1 fusion and identify it as a classic ES. Although the question of whether CHP-100 was a virtual and never existing cell line from the beginning is now clarified, the results of all relevant publications should be considered questionable. Neither the time of the cross-contamination event with IMR-32 is known nor was the final classification as a model for Ewing family of tumors available with an associated short tandem repeat profile. After a long road of errors and confusion, authentic CHP-100 is now characterized as a type II EWSR1-FLI1 fusion model 44 years after its establishment.