A variant-dependent molecular clock with anomalous diffusion models SARS-CoV-2 evolution in humans.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans has been monitored at an unprecedented level due to the public health crisis, yet the stochastic dynamics underlying such a process is dubious. Here, considering the number of acquired mutations as the displacement of the viral particle from the origin, we performed biostatistical analyses from numerous whole genome sequences on the basis of a time-dependent probabilistic mathematical model. We showed that a model with a constant variant-dependent evolution rate and nonlinear mutational variance with time (i.e., anomalous diffusion) explained the SARS-CoV-2 evolutionary motion in humans during the first 120 wk of the pandemic in the United Kingdom. In particular, we found subdiffusion patterns for the Primal, Alpha, and Omicron variants but a weak superdiffusion pattern for the Delta variant. Our findings indicate that non-Brownian evolutionary motions occur in nature, thereby providing insight for viral phylodynamics.

A new age of precision gene therapy.

Gene therapy has become a clinical reality as market-approved advanced therapy medicinal products for the treatment of distinct monogenetic diseases and B-cell malignancies. This Therapeutic Review aims to explain how progress in genome editing technologies offers the possibility to expand both therapeutic options and the types of diseases that will become treatable. To frame these impressive advances in the context of modern medicine, we incorporate examples from human clinical trials into our discussion on how genome editing will complement currently available strategies in gene therapy, which still mainly rely on gene addition strategies. Furthermore, safety considerations and ethical implications, including the issue of accessibility, are addressed as these crucial parameters will define the impact that gene therapy in general and genome editing in particular will have on how we treat patients in the near future.

A human genome editing-based MLL::AF4 ALL model recapitulates key cellular and molecular leukemogenic features.

Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.

Unlocking Photocatalytic Activity of Acridinium Salts by Anion-Binding Co-Catalysis.

The in situ generation of active photoredox organic catalysts upon anion-binding co-catalysis by making use of the ionic nature of common photosensitizers is reported. Hence, the merge of anion-binding and photocatalysis permitted the modulation of the photocatalytic activity of simple acridinium halide salts, building an effective anion-binding - photoredox ion pair complex able to promote a variety of visible light driven transformations, such as anti-Markovnikov addition to olefins, Diels-Alder and the desilylative C-C bond forming reactions. Anion-binding studies, together with steady-state and time-resolved spectroscopy analysis, supported the postulated ion pair formation between the thiourea hydrogen-bond donor organocatalyst and the acridinium salt, which proved essential for unlocking the photocatalytic activity of the photosensitizer.

Continuing Low Awareness and Use of Pre-exposure Prophylaxis (PrEP) for HIV among People Who Inject Drugs (PWID), San Francisco, 2022.

Clinical trials provide evidence that pre-exposure prophylaxis (PrEP) prevents HIV acquisition including through sharing of injection equipment among people who inject drugs (PWID). However, uptake among many populations at risk for HIV has been slow, particularly among PWID. We examined data from the National HIV Behavioral Surveillance (NHBS) from San Francisco in 2022 to measure PrEP uptake and identify factors associated with PrEP awareness among PWID. Of 479 PWID with HIV-negative or unknown HIV status, 54.9% were aware of PrEP, 5.9% had discussed PrEP with a healthcare provider, and 1.5% had used PrEP in the past year. Lack of PrEP awareness was associated with being age 50 years and older (adjusted odds ratio [aOR] 0.40, 95% CI 0.27-0.60), being men who have sex with women (vs. men who have sex with men, aOR 0.47, 95% CI 0.24-0.92), having a disability (aOR 0.58, 95% CI 0.35-0.95), using heroin as their most frequently injected drug (aOR 0.51, 95% CI, 0.34-0.78), not having tested for HIV, HCV, or an STD in the past year (aOR 0.43, 95% CI 0.28-0.64), and not having access to new sterile needles in the past year (aOR 0.28, 95%CI 0.08-1.00). We found negligible change in the awareness and uptake of PrEP among PWID since previously measured in NHBS in 2018. Low PrEP use among PWID may be addressed by increasing provider discussion of PrEP with their PWID patients and clients during routine care, expanding testing for injection-related infections among PWID, and integrating PrEP access into harm reduction programs.

Disparities in HIV Testing and PrEP Awareness for Black Women who Inject Drugs in San Francisco, California.

HIV disproportionately affects Black/African Americans (AA), while PrEP is under-utilized by Black/AA, women, and people who inject drugs (PWID). In San Francisco, California's National HIV Behavioral Surveillance among PWID in 2022, Black/AA women were the least likely to be tested for HIV among all groups by sex and race/ethnicity and the least likely to be aware of PrEP among women. Yet, Black/AA women were no less likely to see a healthcare provider in the last year. Data suggest that providers' failure to discuss and address HIV risk with Black/AA female PWID is a major barrier to accessing effective care and prevention. El VIH afecta de manera desproporcionada a Black/afroamericanos (AA), mientras que la PrEP está infrautilizada por los Black/AA, las mujeres y las personas que se inyectan drogas (PWID). En la National HIV Behavioral Surveillance de PWID de San Francisco, California en 2022, las mujeres Black/AA eran las que menos probabilidades tenían de someterse a la prueba del VIH entre todos los grupos por sexo y raza/etnia y las que menos probabilidades tenían de conocer la PrEP entre las mujeres. Sin embargo, las mujeres Black/AA no tenían menos probabilidades de acudir a un profesional sanitario en el último año. Los datos sugieren que el hecho de que los proveedores no hablen ni aborden el riesgo de VIH con las PWID de raza Black/AA es un obstáculo importante para acceder a una atención y prevención eficaces.

Structural basis of direct and inverted DNA sequence repeat recognition by helix-turn-helix transcription factors.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix-turn-helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.

ddPCR Overcomes the CRISPR-Cas13a-Based Technique for the Detection of the BRAF p.V600E Mutation in Liquid Biopsies.

The isolation of circulating tumoral DNA (ctDNA) present in the bloodstream brings about the opportunity to detect genomic aberrations from the tumor of origin. However, the low amounts of ctDNA present in liquid biopsy samples makes the development of highly sensitive techniques necessary to detect targetable mutations for the diagnosis, prognosis, and monitoring of cancer patients. Here, we employ standard genomic DNA (gDNA) and eight liquid biopsy samples from different cancer patients to examine the newly described CRISPR-Cas13a-based technology in the detection of the BRAF p.V600E actionable point mutation and appraise its diagnostic capacity with two PCR-based techniques: quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR). Regardless of its lower specificity compared to the qPCR and ddPCR techniques, the CRISPR-Cas13a-guided complex was able to detect inputs as low as 10 pM. Even though the PCR-based techniques have similar target limits of detection (LoDs), only the ddPCR achieved a 0.1% variant allele frequency (VAF) detection with elevated reproducibility, thus standing out as the most powerful and suitable tool for clinical diagnosis purposes. Our results also demonstrate how the CRISPR-Cas13a can detect low amounts of the target of interest, but its base-pair specificity failed in the detection of actionable point mutations at a low VAF; therefore, the ddPCR is still the most powerful and suitable technique for these purposes.

[Recommendations based on the management of patients with Crohn disease in the Peruvian context. Position paper of the Association for the Study of the Intestine (ASPEIN)]. / Recomendaciones basadas en el manejo de pacientes con diagnóstico de enfermedad de Crohn en el contexto peruano. Documento de posición de la Asociación Peruana para el Estudio del Intestino (ASPEIN).

Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract whose etiology is unknown, which can transmurally affect any segment of the intestine and/or the perineal region. Worldwide, the incidence of inflammatory bowel disease has increased in recent decades, and the same upward trend can be seen in South America. At national level, there are no official data, however, the increase in the number of publications in the last 20 years confirms this upward trend. Crohn's disease is a forgotten disease and does not have implemented clinical guidelines based on evidence that contribute to clinicians in decision making. In this sense, the Peruvian Association for the Study of the Intestine considers the preparation of this document relevant and timely. clinical contextualized for Peru.

Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement.

Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.

Effective Formation of New C(sp2 )-S Bonds via Photoactivation of Alkylamine-based Electron Donor-Acceptor Complexes.

A novel visible light promoted formation of CAryl- S bonds through electron donor-acceptor (EDA) complexes of alkylamines with 5- and 6-membered (hetero)arene halides is presented. This represents the first EDA-based thiolation method not relying on π-π or a thiolate-anion-π interactions and provides a facile access to heteroarene radicals, which can be suitably trapped by disulfide derivatives to form the corresponding versatile arylsulfides. Mechanistic investigations on the aspects of the whole process were conducted by spectroscopic measurements, demonstrating the hypothesized EDA complex formation. Moreover, the strength of this method has been proven by a gram-scale synthesis of thiolated products and the late-stage derivatization of an anticoagulant drug.

Dual-mode colorimetric/fluorescent chemosensor for Cu2+/Zn2+ and fingerprint imaging based on rhodamine ethylenediamine bis(triazolyl silsesquioxane).

A novel dual functional and visual rhodamine ethylenediamine bis(triazolyl silsesquioxane) (RBS) chemosensor was successfully synthesized using "click" chemistry. The results have unambiguously demonstrated that RBS can act in fluorescent and colorimetric sensing of Cu2+ and Zn2+ by their respective coordination with triazole structures and, more importantly, it has also been found that triazole-amide of RBS could turn on chelation-enhanced fluorescence (CHEF) of Cu2+. Remarkably, the addition of Cu2+ triggered an enhanced fluorescent emission by 63.3-fold (ϕF = 0.41), while Zn2+ enhanced it 48.3-fold (ϕF = 0.29) relative to the original RBS (ϕF = 0.006) in acetonitrile (MeCN) solvent. The fluorescent limit of detection for Cu2+ and Zn2+ is similar and fall within 3.0 nM, while under colorimetric sensing the responses were 2.14 × 10-8 and 4.0 × 10-8 mol L-1, respectively. Moreover, the effective sensing profile of RBS and extended applications of RBS-Cu2+ and RBS-Zn2+ for fingerprinting detection and imaging were observed with adequate sensitivity, stability and legibility under the dual visual responses.

A new green-to-blue upconversion system with efficient photoredox catalytic properties.

The design and development of new triplet-triplet annihilation upconversion (TTA-UC) systems combining triplet sensitizers with acceptor compounds have attracted considerable interest. In this vein, sensitizers made from purely organic dyes rather than transition-metal complexes appear to be more convenient from an environmental point of view. BODIPYs are a very well-known class of dyes with applications in a widespread range of scientific areas. Owing to the versatility of BODIPYs, we present herein a new asymmetric BODIPY with excellent photophysical properties to be used as an appropriate sensitizer in a bimolecular TTA-UC system. Detailed spectroscopic measurements demonstrated the ability of this new design to sensitize TTA-UC by combination with a suitable acceptor such as 2,5,8,11-tetra-tert-butylperylene (TBPe), allowing a successful conversion of green to blue light. The singlet-excited TBPe so obtained is capable of activating aryl chlorides reductively which initiated the functionalization of N-methylpyrrole (Meerwein-type arylation) and formation of both substituted triarylethylenes (Mizoroki-Heck reaction) and heteroarene phosphonates (photo-Arbuzov reaction). Product yields reveal that our TTA-UC system behaved as a highly efficient photocatalytic entity.

A novel and efficient tandem CD19- and CD22-directed CAR for B cell ALL.

CD19-directed chimeric antigen receptor (CAR) T cells have yielded impressive response rates in refractory/relapse B cell acute lymphoblastic leukemia (B-ALL); however, most patients ultimately relapse due to poor CAR T cell persistence or resistance of either CD19+ or CD19- B-ALL clones. CD22 is a pan-B marker whose expression is maintained in both CD19+ and CD19- relapses. CD22-CAR T cells have been clinically used in B-ALL patients, although relapse also occurs. T cells engineered with a tandem CAR (Tan-CAR) containing in a single construct both CD19 and CD22 scFvs may be advantageous in achieving higher remission rates and/or preventing antigen loss. We have generated and functionally validated using cutting-edge assays a 4-1BB-based CD22/CD19 Tan-CAR using in-house-developed novel CD19 and CD22 scFvs. Tan-CAR-expressing T cells showed similar in vitro expansion to CD19-CAR T cells with no increase in tonic signaling. CRISPR-Cas9-edited B-ALL cells confirmed the bispecificity of the Tan-CAR. Tan-CAR was as efficient as CD19-CAR in vitro and in vivo using B-ALL cell lines, patient samples, and patient-derived xenografts (PDXs). Strikingly, the robust antileukemic activity of the Tan-CAR was slightly more effective in controlling the disease in long-term follow-up PDX models. This Tan-CAR construct warrants a clinical appraisal to test whether simultaneous targeting of CD19 and CD22 enhances leukemia eradication and reduces/delays relapse rates and antigen loss.

A faecal microbiota signature with high specificity for pancreatic cancer.

BACKGROUND: Recent evidence suggests a role for the microbiome in pancreatic ductal adenocarcinoma (PDAC) aetiology and progression. OBJECTIVE: To explore the faecal and salivary microbiota as potential diagnostic biomarkers. METHODS: We applied shotgun metagenomic and 16S rRNA amplicon sequencing to samples from a Spanish case-control study (n=136), including 57 cases, 50 controls, and 29 patients with chronic pancreatitis in the discovery phase, and from a German case-control study (n=76), in the validation phase. RESULTS: Faecal metagenomic classifiers performed much better than saliva-based classifiers and identified patients with PDAC with an accuracy of up to 0.84 area under the receiver operating characteristic curve (AUROC) based on a set of 27 microbial species, with consistent accuracy across early and late disease stages. Performance further improved to up to 0.94 AUROC when we combined our microbiome-based predictions with serum levels of carbohydrate antigen (CA) 19-9, the only current non-invasive, Food and Drug Administration approved, low specificity PDAC diagnostic biomarker. Furthermore, a microbiota-based classification model confined to PDAC-enriched species was highly disease-specific when validated against 25 publicly available metagenomic study populations for various health conditions (n=5792). Both microbiome-based models had a high prediction accuracy on a German validation population (n=76). Several faecal PDAC marker species were detectable in pancreatic tumour and non-tumour tissue using 16S rRNA sequencing and fluorescence in situ hybridisation. CONCLUSION: Taken together, our results indicate that non-invasive, robust and specific faecal microbiota-based screening for the early detection of PDAC is feasible.

Photoredox catalysis powered by triplet fusion upconversion: arylation of heteroarenes.

In this work, the feasibility of triplet fusion upconversion (TFU, also named triplet-triplet annihilation upconversion) technology for the functionalization (arylation) of furans and thiophenes has been successfully proven. Activation of aryl halides by TFU leads to generation of aryl radical intermediates; trapping of the latter by the corresponding heteroarenes, which act as nucleophiles, affords the final coupling products. Advantages of this photoredox catalytic method include the use of very mild conditions (visible light, standard conditions), employment of commercially available reactants and low-loading metal-free photocatalysts, absence of any sacrificial agent (additive) in the medium and short irradiation times. The involvement of the high energetic delayed fluorescence in the reaction mechanism has been evidenced by quenching studies, whereas the two-photon nature of this photoredox arylation of furans and thiophenes has been manifested by the dependence on the energy source power. Finally, the scaling-up conditions have been gratifyingly afforded by a continuous-flow device.

Human-specific changes in two functional enhancers of FOXP2.

FOXP2 is a gene involved in language development and function. Neanderthals and humans share the same coding region of the gene, although the formers are thought to have exhibited less sophisticated language abilities. In this paper, we report on several human-specific changes in two functional enhancers of FOXP2. Two of these variants are located within the binding sites for the transcription factors POLR2A and SMARCC1, respectively. Interestingly, SMARCC1 is involved in brain development and vitamin D metabolism. We hypothesize that the human specific change in this position might have resulted in a different regulation pattern of FOXP2 expression in our species compared to extinct hominins, with a potential impact on our language abilities.

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress.

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

CRISPR Approaches for the Diagnosis of Human Diseases.

CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.

[Clinical practice guideline for Diagnosis and management of cholelithiasis, cholecystitis and choledocholithiasis for the Peruvian Social Security (EsSalud)]. / Guía de práctica clínica para el diagnóstico y manejo de la colelitiasis, colecistitis aguda y coledocolitiasis en el Seguro Social del Perú (EsSalud).

INTRODUCTION: This article summarizes the clinical practice guide (CPG) for the diagnosis and management of cholelithiasis, acute cholecystitis and choledocholithiasis in the Peruvian Social Security (EsSalud). OBJECTIVE: To provide clinical recommendations based on evidence for the management of patients with cholelithiasis, acute cholecystitis and choledocholithiasis in EsSalud. METHODS: a guideline task force (GTF) was formed with internists, general surgeons, gastroenterologists, and methodologists. The group proposed 10 clinical questions to be answered in this Clinical practice guideline (CPG). Systematic searches of preview reviews were performed and when it was necessary, primary studies from PubMed and CENTRAL during 2017 were reviewed. The evidence was selected aiming to answer each proposed question. Certainty of evidence was evaluated using Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology. In periodical work sessions, the group used GRADE methodology for reviewing the evidence and formulating recommendations, good clinical practice items and three flowcharts for diagnosis and treatment. Finally, the CPG was approved by Resolution Nº 046-IETSI-ESSALUD-2017. RESULTS: This CPG approached 10 clinical questions divided into two topics: diagnosis and management. Based on these questions; one strong recommendation, five weak recommendations, and 17 good clinical practice items and three flowcharts were formulated. CONCLUSION: This paper abstracts the methodology and evidence-based conclusions of the CPG for diagnosis and management of cholelithiasis, acute cholecystitis and choledocholithiasis in EsSalud.