Management of newborns and healthcare workers exposed to isoniazid-resistant congenital tuberculosis in the neonatal intensive care unit.

BACKGROUND: Management of newborns and healthcare workers (HCWs) exposed to congenital tuberculosis (TB) in the neonatal intensive care unit (NICU) has been reported rarely. AIM: To outline a contact investigation process for individuals exposed to congenital TB in the NICU and investigate nosocomial transmission. Additionally, to assess the efficacy and safety of window prophylaxis in exposed newborns. METHODS: A baby, born at a gestational age of 28 + 1 weeks, was diagnosed with isoniazid-resistant congenital TB on the 39th day of admission to the level IV NICU. Newborns and HCWs exposed cumulatively for ≥8 h underwent contact investigation and follow-up for a year. FINDINGS: Eighty-two newborns underwent contact investigation. All newborns displayed normal chest X-rays, and 42 hospitalized newborns tested negative for acid-fast bacilli stain and Xpert® MTB/RIF assay in their endotracheal sputum or gastric juices. Eighty received window prophylaxis: six of 75 on rifampin experienced mild adverse events, and none of the five on levofloxacin. After 12 weeks, five (6.1%) had a positive tuberculin skin test, all of whom had already received the Bacillus Calmette-Guérin vaccine and tested negative on TB interferon-gamma releasing assay. Of 119 exposed HCWs, three (2.5%) were diagnosed with latent TB infection and completed a four-month rifampin therapy. There was no active TB disease among exposed newborns and HCWs during a one-year follow-up. CONCLUSION: Timely diagnosis of congenital TB is crucial for minimizing transmission among exposed neonates and HCWs in the NICU setting. In cases of isoniazid-resistant index patients, even premature newborns may consider the use of rifampin or levofloxacin for window prophylaxis.

Compliance with new drug use and the effect of discrepant drug susceptibility testing on MDR/RR-TB treatment.

BACKGROUND: Following the WHO???s announcement in 2018, the use of new drugs was recommended for all patients with multidrug-resistant TB (MDR-TB) in Korea. This study aimed to evaluate adherence to new anti-TB drug regimens and implementation of molecular drug susceptibility testing (mDST) in Korea.METHODS: Nationwide, 560 patients were reported as having MDR-TB in 2021. The implementation of mDST and new anti-TB drug use were analysed. The discrepancy between mDST and phenotypic DST (pDST) results and their implications on the use of new anti-TB drugs were also analysed. The use of novel anti-TB drugs has been approved by the National TB Expert Committee.RESULTS: The non-adherence rate in MDR-TB patients was 14.3%. The mDST implementation rate was 96.1%. Of the 459 patients who underwent both mDST and pDST, the discordance rate for rifampicin (RIF) resistance was 22.6% (n = 104), of which 72.1% (n = 75) were resistant on mDST but susceptible on pDST. The discrepancy in mDST and pDST results related to RIF resistance was found to be the main cause of non-adherence to new drug regimen.CONCLUSION: Comprehensive training on how to interpret conflicting results between mDST and pDST could enhance the utilisation of new drugs in the treatment of MDR/RIF-resistant TB.

Identification of 27 allele-specific regulatory variants in Parkinson's disease using a massively parallel reporter assay.

Genome wide association studies (GWAS) have identified a number of genomic loci that are associated with Parkinson's disease (PD) risk. However, the majority of these variants lie in non-coding regions, and thus the mechanisms by which they influence disease development, and/or potential subtypes, remain largely elusive. To address this, we used a massively parallel reporter assay (MPRA) to screen the regulatory function of 5254 variants that have a known or putative connection to PD. We identified 138 loci with enhancer activity, of which 27 exhibited allele-specific regulatory activity in HEK293 cells. The identified regulatory variant(s) typically did not match the original tag variant within the PD associated locus, supporting the need for deeper exploration of these loci. The existence of allele specific transcriptional impacts within HEK293 cells, confirms that at least a subset of the PD associated regions mark functional gene regulatory elements. Future functional studies that confirm the putative targets of the empirically verified regulatory variants will be crucial for gaining a greater understanding of how gene regulatory network(s) modulate PD risk.

Development and characterisation of a novel inhibitory anti-GH monoclonal antibody.

Excess growth hormone (GH) has been implicated in multiple cancer types and there is increasing interest in the development of therapeutic inhibitors targeting GH-GH receptor (GHR) signalling. Here we describe a panel of anti-GH monoclonal antibodies (mAbs) generated using a hybridoma approach and identify two novel inhibitory mAbs (1-8-2 and 1-46-3) that neutralised GH signalling. mAbs 1-8-2 and 1-46-3 exhibited strong inhibitory activity against GH-dependent cell growth in a Ba/F3-GHR cell viability assay, with EC50 values of 1.00 ± 0.27 and 0.5 ± 0.1 µg/mL, respectively. Cross-reactivity with the human placental hormones, placental lactogen (PL) and placental GH, was observed by ELISA, but neither antibody cross-reacted with mouse GH or human prolactin (PRL). mAb 1-8-2 had a binding affinity for GH of KD 0.62 ± 0.5 nM, while mAb 1-46-3 had a KD of 2.68 ± 0.53 nM, as determined by bio-layer interferometry. mAb 1-46-3 inhibited GH-dependent signal transduction in T-47D and LNCaP cancer cell lines and reduced GH-dependent cell growth and migration in the breast cancer cell line T-47D. mAb 1-46-3 inhibited T-47D cell viability more effectively than the GHR antagonist B2036. In conclusion, we describe two novel inhibitory anti-GH mAbs and provide in vitro evidence supporting development of these entities as anti-cancer therapeutics.

Treatment outcome, recurrence and safety of multidrug-resistant TB treated with low-dose linezolid.

BACKGROUND: Linezolid (LZD) is a key treatment option for patients with multidrug-resistant/rifampicin-resistant TB (MDR/RR-TB). We investigated the long-term treatment outcomes and safety of MDR/RR-TB treatment using low-dose LZD.METHODS: Medical records of patients with MDR/RR-TB treated with LZD ≥4 weeks between 2004 and 2018 at the Asan Medical Center, Seoul, Republic of Korea, were reviewed. Standard-dose and low-dose LZD groups were defined as patients initially administered LZD ≥600 mg/day or 300 mg/day, respectively.RESULTS: Among 94 patients, 65 were included in the low-dose LZD group; mean age was 43.1 ± 15.6 years, 53 (56.4%) were men and 77 (83.7%) were resistant to fluoroquinolone. The low-dose LZD group showed features of less severe disease, such as limited MDR-TB history and less severe radiological findings. There was no difference in treatment outcomes, relapse and safety between groups. In the low-dose LZD group, 54 (83.1%) succeeded treatment, of whom 48 (88.9%) were followed-up for a median of 38 months; there was no recurrence. Adverse drug reactions were reported in 41 (63.1%); peripheral neuropathy was most frequently reported (n = 31, 47.7%), while myelosuppression was reported in 12 (18.5%).CONCLUSION: Low-dose LZD in selected patients with less severe disease is both effective in the long-term and safe for the treatment of MDR/RR-TB.

What over 1,000,000 participants tell us about online research protocols.

With the ever-increasing adoption of tools for online research, for the first time we have visibility on macro-level trends in research that were previously unattainable. However, until now this data has been siloed within company databases and unavailable to researchers. Between them, the online study creation and hosting tool Gorilla Experiment Builder and the recruitment platform Prolific hold metadata gleaned from millions of participants and over half a million studies. We analyzed a subset of this data (over 1 million participants and half a million studies) to reveal critical information about the current state of the online research landscape that researchers can use to inform their own study planning and execution. We analyzed this data to discover basic benchmarking statistics about online research that all researchers conducting their work online may be interested to know. In doing so, we identified insights related to: the typical study length, average completion rates within studies, the most frequent sample sizes, the most popular participant filters, and gross participant activity levels. We present this data in the hope that it can be used to inform research choices going forward and provide a snapshot of the current state of online research.

Growth hormone receptor agonists and antagonists: From protein expression and purification to long-acting formulations.

Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to the development of long-acting formulations required to improve the circulating half-life of the drug. In this review, we summarize methodologies and approaches used for making and purifying recombinant GH and GHA proteins, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed.

Targeting growth hormone in cancer: future perspectives.

Decades of published research support a role for growth hormone (GH) in cancer. Accordingly, there is increasing interest in targeting GH in oncology, with GH antagonists exhibiting efficacy in xenograft studies as single agents and in combination with anticancer therapy or radiation. Here we discuss challenges associated with using growth hormone receptor (GHR) antagonists in preclinical models and considerations for translation, such as the identification of predictive biomarkers for selecting patients and for monitoring drug efficacy. Ongoing research will determine whether suppressing GH signalling pharmacologically will also reduce the risk of developing cancer. An increase in GH-targeted drugs in preclinical development will ultimately provide new tools to test anticancer efficacy of blocking the GH signalling pathway.

The growth hormone receptor interacts with transcriptional regulator HMGN1 upon GH-induced nuclear translocation.

Growth hormone (GH) actions are mediated through binding to its cell-surface receptor, the GH receptor (GHR), with consequent activation of downstream signalling. However, nuclear GHR localisation has also been observed and is associated with increased cancer cell proliferation. Here we investigated the functional implications of nuclear translocation of the GHR in the human endometrial cancer cell-line, RL95-2, and human mammary epithelial cell-line, MCF-10A. We found that following GH treatment, the GHR rapidly translocates to the nucleus, with maximal localisation at 5-10 min. Combined immunoprecipitation-mass spectrometry analysis of RL95-2 whole cell lysates identified 40 novel GHR binding partners, including the transcriptional regulator, HMGN1. Moreover, microarray analysis demonstrated that the gene targets of HMGN1 were differentially expressed following GH treatment, and co-immunoprecipitation showed that HMGN1 associates with the GHR in the nucleus. Therefore, our results suggest that GHR nuclear translocation might mediate GH actions via interaction with chromatin factors that then drive changes in specific downstream transcriptional programs.

Interpretation of the role of germline and somatic non-coding mutations in cancer: expression and chromatin conformation informed analysis.

BACKGROUND: There has been extensive scrutiny of cancer driving mutations within the exome (especially amino acid altering mutations) as these are more likely to have a clear impact on protein functions, and thus on cell biology. However, this has come at the neglect of systematic identification of regulatory (non-coding) variants, which have recently been identified as putative somatic drivers and key germline risk factors for cancer development. Comprehensive understanding of non-coding mutations requires understanding their role in the disruption of regulatory elements, which then disrupt key biological functions such as gene expression. MAIN BODY: We describe how advancements in sequencing technologies have led to the identification of a large number of non-coding mutations with uncharacterized biological significance. We summarize the strategies that have been developed to interpret and prioritize the biological mechanisms impacted by non-coding mutations, focusing on recent annotation of cancer non-coding variants utilizing chromatin states, eQTLs, and chromatin conformation data. CONCLUSION: We believe that a better understanding of how to apply different regulatory data types into the study of non-coding mutations will enhance the discovery of novel mechanisms driving cancer.

Identifying the lungs as a susceptible site for allele-specific regulatory changes associated with type 1 diabetes risk.

Type 1 diabetes (T1D) etiology is complex. We developed a machine learning approach that ranked the tissue-specific transcription regulatory effects for T1D SNPs and estimated their relative contributions to conversion to T1D by integrating case and control genotypes (Wellcome Trust Case Control Consortium and UK Biobank) with tissue-specific expression quantitative trait loci (eQTL) data. Here we show an eQTL (rs6679677) associated with changes to AP4B1-AS1 transcript levels in lung tissue makes the largest gene regulatory contribution to the risk of T1D development. Luciferase reporter assays confirmed allele-specific enhancer activity for the rs6679677 tagged locus in lung epithelial cells (i.e. A549 cells; C > A reduces expression, p = 0.005). Our results identify tissue-specific eQTLs for SNPs associated with T1D. The strongest tissue-specific eQTL effects were in the lung and may help explain associations between respiratory infections and risk of islet autoantibody seroconversion in young children.

Who is a credible source of preventive advice? An experimental vignette study of general public attitudes towards role expansion in health and social care.

OBJECTIVES: To investigate the general public's source credibility attitudes towards health and social care professionals when giving advice associated with their 'traditional role' versus an 'expanded health behaviour change' role, to facilitate the implementation of the health behaviour change agenda. DESIGN: A 3x3 experimental between-subjects vignette questionnaire study with nine scenarios in which a general practitioner (GP), health visitor, or firefighter offered advice on either stopping smoking, preventing cot death, or fire safety. Combinations were either congruent with a traditional role (e.g., health visitor and cot death) or an expanded role (e.g., firefighter and stopping smoking). METHODS: Adults were recruited from metropolitan locations in northern England. Participants were randomized to one scenario and complete a validated 18-item source credibility questionnaire. Factor analysis explored source credibility components; ratings for traditional and expanded role scenarios were compared using Mann-Whitney tests. RESULTS: 369 participants completed the questionnaire (49.3% women, 64% White British, age range: 16-83). Factor analysis confirmed three source credibility dimensions: competence, caring, and trustworthiness. Ratings were generally high across professions and scenarios; participants rated professionals as significantly more 'competent' where scenarios related to their traditional roles than expanded roles (U  9778.5, p  <.001) but equally as caring (U = 14467.5, p< .485) and trustworthy (U  14250.5, p  .348). CONCLUSIONS: GPs, health visitors, and firefighters were all perceived as credible sources of health behaviour change advice, but may be viewed as 'less competent' sources of messages associated with an expanded job role. Effective professional training and public engagement regarding the role expansion agenda are needed to support policy implementation. Statement of Contribution What is already known on this subject? Source credibility surrounds people's perceptions of the source of advice and includes competence, caring, and trustworthiness dimensions. This may have an important influence on people's attitudes and behaviour, especially when messages are complex or emotive. A wide range of public sector workers is now expected to routinely offer preventive health and safety advice, as part of role expansion. What does this study add? This experimental survey study compared source credibility perceptions of GPs, health visitors, and firefighters giving advice on topics associated with a traditional or expanded role. Professionals were perceived as less 'competent' when giving advice on 'expanded role' topics, such as a firefighter discussing smoking, with a small reduction in source credibility. This highlights that source credibility may be specific to professional identities. Policymakers may need to explore this further as part of implementing role expansion for prevention and self-management in health and social care.

Realistic precision and accuracy of online experiment platforms, web browsers, and devices.

Due to increasing ease of use and ability to quickly collect large samples, online behavioural research is currently booming. With this popularity, it is important that researchers are aware of who online participants are, and what devices and software they use to access experiments. While it is somewhat obvious that these factors can impact data quality, the magnitude of the problem remains unclear. To understand how these characteristics impact experiment presentation and data quality, we performed a battery of automated tests on a number of realistic set-ups. We investigated how different web-building platforms (Gorilla v.20190828, jsPsych v6.0.5, Lab.js v19.1.0, and psychoJS/PsychoPy3 v3.1.5), browsers (Chrome, Edge, Firefox, and Safari), and operating systems (macOS and Windows 10) impact display time across 30 different frame durations for each software combination. We then employed a robot actuator in realistic set-ups to measure response recording across the aforementioned platforms, and between different keyboard types (desktop and integrated laptop). Finally, we analysed data from over 200,000 participants on their demographics, technology, and software to provide context to our findings. We found that modern web platforms provide reasonable accuracy and precision for display duration and manual response time, and that no single platform stands out as the best in all features and conditions. In addition, our online participant analysis shows what equipment they are likely to use.

Enhanced Bioactivity of a Human GHR Antagonist Generated by Solid-Phase Site-Specific PEGylation.

Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036-S144C was expressed in Escherichia coli, purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036-S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC50 values for the 20, 30, and 40 kDa mPEG maleimide B2036-S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo.

Genetic Code Expansion Enables Site-Specific PEGylation of a Human Growth Hormone Receptor Antagonist through Click Chemistry.

Regulation of human growth hormone (GH) signaling has important applications in the remediation of several diseases including acromegaly and cancer. Growth hormone receptor (GHR) antagonists currently provide the most effective means for suppression of GH signaling. However, these small 22 kDa recombinantly engineered GH analogues exhibit short plasma circulation times. To improve clinical viability, between four and six molecules of 5 kDa poly(ethylene glycol) (PEG) are nonspecifically conjugated to the nine amines of the GHR antagonist designated as B2036 in the FDA-approved therapeutic pegvisomant. PEGylation increases the molecular weight of B2036 and considerably extends its circulation time, but also dramatically reduces its bioactivity, contributing to high dosing requirements and increased cost. As an alternative to nonspecific PEGylation, we report the use of genetic code expansion technology to site-specifically incorporate the unnatural amino acid propargyl tyrosine (pglY) into B2036 with the goal of producing site-specific protein-polymer conjugates. Substitution of tyrosine 35 with pglY yielded a B2036 variant containing an alkyne functional group without compromising bioactivity, as verified by a cellular assay. Subsequent conjugation of 5, 10, and 20 kDa azide-containing PEGs via the copper-catalyzed click reaction yielded high purity, site-specific conjugates with >89% conjugation efficiencies. Site-specific attachment of PEG to B2036 is associated with substantially improved in vitro bioactivity values compared to pegvisomant, with an inverse relationship between polymer size and activity observed. Notably, the B2036-20 kDa PEG conjugate has a molecular weight comparable to pegvisomant, while exhibiting a 12.5 fold improvement in half-maximal inhibitory concentration in GHR-expressing Ba/F3 cells (103.3 nM vs 1289 nM). We expect that this straightforward route to achieve site-specific GHR antagonists will be useful for GH signal regulation.

Evaluation of breath, plasma, and urinary markers of lactose malabsorption to diagnose lactase non-persistence following lactose or milk ingestion.

BACKGROUND: Adult lactase non-persistence (LNP) is due to low lactase expression, resulting in lactose malabsorption (LM). LNP is a genetic trait, but is typically determined by LM markers including breath H2, blood glucose, and urinary galactose after a lactose tolerance test. Known validity of these markers using milk is limited, despite being common practice. Compositional variation, such as ß-casein variants, in milk may impact diagnostic efficacy. This study aimed to evaluate the diagnostic accuracy to detect LNP using these commonly measured LM markers after both lactose and milk challenges. METHODS: Fourty healthy young women were challenged with 50 g lactose then randomized for separate cross-over visits to ingest 750 mL milk (37.5 g lactose) as conventional (both A1 and A2 ß-casein) and A1 ß-casein-free (a2 Milk™) milk. Blood, breath and urine were collected prior to and up to 3 h following each challenge. The presence of C/T13910 and G/A22018 polymorphisms, determined by restriction fragment length polymorphism, was used as the diagnostic reference for LNP. RESULTS: Genetic testing identified 14 out of 40 subjects as having LNP (C/C13910 and G/G22018). All three LM markers (breath H2, plasma glucose and urinary galactose/creatinine) discriminated between lactase persistence (LP) and LNP following lactose challenge with an area under the receiver operating characteristic (ROC) curve (AUC) of 1.00, 0.75 and 0.73, respectively. Plasma glucose and urinary galactose/creatinine were unreliable (AUC < 0.70) after milk ingestion. The specificity of breath H2 remained high (100%) when milk was used, but sensitivity was reduced with conventional (92.9%) and a2 Milk™ (78.6%) compared to lactose (sensitivities adjusted for lactose content). The breath H2 optimal cut-off value was lower with a2 Milk™ (13 ppm) than conventional milk (21 ppm). Using existing literature cut-off values the sensitivity and specificity of breath H2 was greater than plasma glucose to detect LNP following lactose challenge whereas values obtained for urinary galactose/creatinine were lower than the existing literature cut-offs. CONCLUSION: This study showed accurate diagnosis of LNP by breath H2 irrespective of the substrate used, although the diagnostic threshold may vary depending on the lactose substrate or the composition of the milk. TRIAL REGISTRATION: ACTRN12616001694404 . Registered prospectively on December 9, 2016.