GRK specificity and Gßγ dependency determines the potential of a GPCR for arrestin-biased agonism.

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent ß-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein ßγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of ß-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gßγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated ß-arrestin2 recruitment to activated receptors. This was independent of the source of free Gßγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, ß-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gßγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of ß-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gßγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands.

Plant synthetic biology as a tool to help eliminate hidden hunger.

Agricultural systems are under increasing pressure from declining environmental conditions, a growing population, and changes in consumer preferences, resulting in widespread malnutrition-related illnesses. Improving plant nutritional content through biotechnology techniques such as synthetic biology is a promising strategy to help combat hidden hunger caused by the lack of affordable and healthy foods in human diets. Production of compounds usually found in animal-rich diets, such as vitamin D or omega-3 fatty acids, has been recently demonstrated in planta. Here, we review recent biotechnological approaches to biofortifying plants with vitamins, minerals, and other metabolites, and summarise synthetic biology advances that offer the opportunity to build on these early biofortification efforts.

Long-Term Safety of Level II Oncoplastic Surgery after Neoadjuvant Treatment for Locally Advanced Breast Cancer: A 20-Year Experience.

Background: Oncoplastic surgery (OPS) reliability in the post-neoadjuvant chemotherapy (NACT) setting is still debated due to weak scientific evidences in such scenarios. Methods: Our analysis aims to report results obtained in a retrospective series of 111 patients consecutively treated with level II OPS after NACT at the Multidisciplinary Breast Center of the Fondazione Policlinico Universitario Agostino Gemelli IRCCS between 1998 and 2018. The surgical endpoints were the mean specimen volume, rates of positive margins (PMR), re-excision (RR), conversion to mastectomy (CMR), and complications (CR). The oncological endpoints were overall survival (OS), disease-free survival (DFS), and local recurrence (LR). To evaluate the impact of NACT on surgical and oncological outcomes at 302 months, we conducted a propensity score matching, pairing patients in post-NACT and upfront surgery groups. Results: The mean sample volume was 390,796 mm3. We registered a 3.6% of PMR, 1.8% RR, 0.9% CMR, 5% CR. The 10-year OS and 10-year DFS with a median follow-up of 88 months (6-302) were 79% and 76%, respectively, with an LR recurrence rate of 5%. The post-NACT group received significantly larger excised volumes and lower PMR. NACT did not affect surgical and oncological outcomes. Conclusions: Level II OPS can be considered a reliable alternative to mastectomy even in the post-NACT setting.

Study Protocol: Pegasus: psychotherapy incorporating horses for 'therapy-resistant' adolescents with autism spectrum disorders, a study with series of randomised, baseline controlled n-of-1 trials.

BACKGROUND: For people with autism spectrum disorder (ASD), daily life can be highly stressful with many unpredictable events that can evoke emotion dysregulation (ED): a strong difficulty with appropriately negative affect regulation. For some of the patients with ASD, treatment as usual does not prove to be effective for ED. They may be at risk of life-long impairment, development of other disorders and loss of motivation for most regular forms of therapy. A highly promising method that may prove effective for therapy-resistant individuals with ASD is Psychotherapy incorporating horses (PIH). PIH uses the interactions of the horse and the patients on the ground and does not include horseriding. While often met with prejudgment and scepticism, reports from parents and therapists as well as a recent systematic review suggest that PIH may have beneficial effects on youths with ASD. Therefore, we examine clinical outcomes both in the short and in the long terms of PIH offered to adolescents with ASD and severe ED despite regular therapy. METHODS: A total of 35 adolescents aged 11-18 years with ASD will receive PIH during 15 sessions once a week with randomization to five different groups differentiating in baseline phase from 2 to 6 weeks. PIH uses horses to promote social awareness and self-awareness as well as relationship management and self-management. The primary outcome is the response to treatment on the Emotion Dysregulation Index (EDI). The secondary outcome measures include ASD symptom severity, quality of life, self-esteem, global and family functioning, and goal attainment. Assessments take place at the baseline (T0), at the end of baseline phase A (T1), after completion of intervention phase B (T2), after the end of post-measurement phase C (T3) and after one year (T4). Qualitative interviews of participants, parents and therapists will be held to reveal facilitators and barriers of PIH and a cost-effectiveness study will be performed. DISCUSSION: This study aims at contributing to clinical practice for adolescents with ASD and persistent emotion regulation problems despite 1.5 year of treatment by offering Psychotherapy incorporating horses in a study with series of randomised, baseline controlled n-of-1 trials. TRIAL REGISTRATION: www. CLINICALTRIALS: gov NCT05200351, December 10th 2021.

Study on the temporal and spatial distribution of Culex mosquitoes in Hanoi, Vietnam.

Arboviruses transmitted by mosquitoes, including Japanese encephalitis virus (JEV), present a substantial global health threat. JEV is transmitted by mosquitoes in the genus Culex, which are common in both urban and rural areas in Vietnam. In 2020, we conducted a 1-year survey of Culex mosquito abundance in urban, suburban, and peri-urban areas of Hanoi using CDC-light traps. Mosquitoes were identified to species and sorted into pools based on species, sex, and trap location. The mosquito pools were also investigated by RT-qPCR for detection of JEV. In total, 4829 mosquitoes were collected over a total of 455 trap-nights, across 13 months. Collected mosquitoes included Culex, Aedes, Anopheles, and Mansonia species. Culex mosquitoes, primarily Cx. quinquefasciatus, predominated, especially in peri-urban areas. Most Culex mosquitoes were caught in the early months of the year. The distribution and abundance of mosquitoes exhibited variations across urban, suburban, and peri-urban sites, emphasizing the influence of environmental factors such as degree of urbanization, temperature and humidity on Culex abundance. No JEV was detected in the mosquito pools. This study establishes baseline knowledge of Culex abundance and temporal variation, which is crucial for understanding the potential for JEV transmission in Hanoi.

X chromosome dosage drives statin-induced dysglycemia and mitochondrial dysfunction.

Statin drugs lower blood cholesterol levels for cardiovascular disease prevention. Women are more likely than men to experience adverse statin effects, particularly new-onset diabetes (NOD) and muscle weakness. Here we find that impaired glucose homeostasis and muscle weakness in statin-treated female mice are associated with reduced levels of the omega-3 fatty acid, docosahexaenoic acid (DHA), impaired redox tone, and reduced mitochondrial respiration. Statin adverse effects are prevented in females by administering fish oil as a source of DHA, by reducing dosage of the X chromosome or the Kdm5c gene, which escapes X chromosome inactivation and is normally expressed at higher levels in females than males. As seen in female mice, we find that women experience more severe reductions than men in DHA levels after statin administration, and that DHA levels are inversely correlated with glucose levels. Furthermore, induced pluripotent stem cells from women who developed NOD exhibit impaired mitochondrial function when treated with statin, whereas cells from men do not. These studies identify X chromosome dosage as a genetic risk factor for statin adverse effects and suggest DHA supplementation as a preventive co-therapy.

Building Better Bridges: Outcomes of a Community-Partnered New School Transition Intervention for Students on the Autism Spectrum.

New school transitions can be challenging for students on the autism spectrum. No published, evidence-based interventions exist to support families and teachers of students transitioning to elementary and secondary school during this critical period. Using Community Partnered Participatory Research, we developed Building Better Bridges (BBB), a caregiver coaching intervention that includes training on effective school communication, educational rights, advocacy, and child preparation strategies. We compared BBB (n = 83) to a module/resources-only comparison (n = 87) in a four-site randomized controlled trial in racially and ethnically diverse, under-resourced communities. In our intent-to-treat analysis, caregivers and teachers in BBB rated students' transitions to the new classroom as more positive, relative to the comparison group. Results suggest this low-cost intervention can improve the transition process for families and students at high risk of poor transitions.

Adjuvant COX inhibition augments STING signaling and cytolytic T cell infiltration in irradiated 4T1 tumors.

Immune therapy is the new frontier of cancer treatment. Therapeutic radiation is a known inducer of immune response and can be limited by immunosuppressive mediators including cyclooxygenase-2 (COX2) that is highly expressed in aggressive triple negative breast cancer (TNBC). A clinical cohort of TNBC tumors revealed poor radiation therapeutic efficacy in tumors expressing high COX2. Herein, we show that radiation combined with adjuvant NSAID (indomethacin) treatment provides a powerful combination to reduce both primary tumor growth and lung metastasis in aggressive 4T1 TNBC tumors, which occurs in part through increased antitumor immune response. Spatial immunological changes including augmented lymphoid infiltration into the tumor epithelium and locally increased cGAS/STING1 and type I IFN gene expression were observed in radiation-indomethacin-treated 4T1 tumors. Thus, radiation and adjuvant NSAID treatment shifts "immune desert phenotypes" toward antitumor M1/TH1 immune mediators in these immunologically challenging tumors. Importantly, radiation-indomethacin combination treatment improved local control of the primary lesion, reduced metastatic burden, and increased median survival when compared with radiation treatment alone. These results show that clinically available NSAIDs can improve radiation therapeutic efficacy through increased antitumor immune response and augmented local generation of cGAS/STING1 and type I IFNs.

Challenging Diagnosis of Streptococcus intermedius-Associated Empyema in an Immunocompetent Adult: A Case Report and Literature Review.

The significance of Streptococcus intermedius in infectious diseases, especially pleural infections, is gaining recognition. While traditional risk factors like dental procedures and immunosuppression remain pivotal in differential diagnosis, there is an emerging recognition of unconventional clinical presentations and risk factors linked to infections by S. intermedius. This shift compels medical professionals to broaden their diagnostic and therapeutic strategies, underscoring the intricate and evolving nature of managing infections associated with this opportunistic bacterium. We describe the case of a 48-year-old immunocompetent woman with untreated hypertension who experienced a 15-day episode of right-sided chest pain, which worsened with a sudden onset of dyspnea, yet her daily activities remained unaffected. Physical examination suggested a pleuropulmonary syndrome due to significant pleural effusion, with a computed tomography (CT) scan of the lungs revealing about 50% effusion on the right side. Laboratory tests indicated elevated inflammatory markers. Ultrasound-guided thoracentesis extracted purulent fluid compatible with empyema, necessitating the placement of a pleural drain and multiple pleural cavity lavages using alteplase, which led to the removal of substantial infected fluid. Culture of the pleural fluid identified S. intermedius, which was pansusceptible. Treatment with intravenous ceftriaxone was administered, resulting in a favorable clinical outcome. This case highlights the critical nature of recognizing atypical clinical presentations and managing complex bacterial infections in the pleural space.

Coexisting Chronic Rhino-Cerebral Mucormycosis and Actinomyces Infection: A Case Report and Review of the Literature.

Fungal rhino-orbital-cerebral infections present significant treatment challenges, especially in immunocompromised individuals, such as those with diabetes. These infections seldom occur with bacterial co-infections, which complicate their management. This report presents the case of a 74-year-old diabetic male with a long-standing history of left malar pain who experienced rhinorrhea, nasal congestion, and confusion. Diagnostic imaging revealed angioinvasive fungal sinusitis, ultimately attributed to chronic mucormycosis (CM) with concurrent Actinomyces infection, a rarely reported occurrence. We employed a comprehensive treatment strategy, which resulted in a successful recovery after 24 days. Although CM is rare, accounting for approximately 5.6% of cases with mucormycosis, it requires thorough diagnostic evaluation and prolonged treatment. The rarity of co-infections like the one we describe underscores the need for an integrated management approach. Histopathological analysis serves as the gold standard for diagnosis, with treatment typically involving surgical and extensive antifungal interventions.

Clinical and neurogenetic characterisation of autosomal recessive RBL2-associated progressive neurodevelopmental disorder.

Retinoblastoma (RB) proteins are highly conserved transcriptional regulators that play important roles during development by regulating cell-cycle gene expression. RBL2 dysfunction has been linked to a severe neurodevelopmental disorder. However, to date, clinical features have only been described in six individuals carrying five biallelic predicted loss of function (pLOF) variants. To define the phenotypic effects of RBL2 mutations in detail, we identified and clinically characterized a cohort of 28 patients from 18 families carrying LOF variants in RBL2 , including fourteen new variants that substantially broaden the molecular spectrum. The clinical presentation of affected individuals is characterized by a range of neurological and developmental abnormalities. Global developmental delay and intellectual disability were uniformly observed, ranging from moderate to profound and involving lack of acquisition of key motor and speech milestones in most patients. Frequent features included postnatal microcephaly, infantile hypotonia, aggressive behaviour, stereotypic movements and non-specific dysmorphic features. Common neuroimaging features were cerebral atrophy, white matter volume loss, corpus callosum hypoplasia and cerebellar atrophy. In parallel, we used the fruit fly, Drosophila melanogaster , to investigate how disruption of the conserved RBL2 orthologueue Rbf impacts nervous system function and development. We found that Drosophila Rbf LOF mutants recapitulate several features of patients harboring RBL2 variants, including alterations in the head and brain morphology reminiscent of microcephaly, and perturbed locomotor behaviour. Surprisingly, in addition to its known role in controlling tissue growth during development, we find that continued Rbf expression is also required in fully differentiated post-mitotic neurons for normal locomotion in Drosophila , and that adult-stage neuronal re-expression of Rbf is sufficient to rescue Rbf mutant locomotor defects. Taken together, this study provides a clinical and experimental basis to understand genotype-phenotype correlations in an RBL2 -linked neurodevelopmental disorder and suggests that restoring RBL2 expression through gene therapy approaches may ameliorate aspects of RBL2 LOF patient symptoms.

Vector competence of Swedish Culex pipiens mosquitoes for Japanese encephalitis virus.

BACKGROUND: Japanese encephalitis virus (JEV) is an emerging mosquito-borne Orthoflavivirus that poses a significant public health risk in many temperate and tropical regions in Asia. Since the climate in some endemic countries is similar to temperate climates observed in Europe, understanding the role of specific mosquito species in the transmission of JEV is essential for predicting and effectively controlling the potential for the introduction and establishment of JEV in Europe. METHODS: This study aimed to investigate the vector competence of colonized Culex pipiens biotype molestus mosquitoes for JEV. The mosquitoes were initially collected from the field in southern Sweden. The mosquitoes were offered a blood meal containing the Nakayama strain of JEV (genotype III), and infection rates, dissemination rates, and transmission rates were evaluated at 14, 21, and 28 days post-feeding. RESULTS: The study revealed that colonized Swedish Cx. pipiens are susceptible to JEV infection, with a stable infection rate of around 10% at all timepoints. However, the virus was only detected in the legs of one mosquito at 21 days post-feeding, and no mosquito saliva contained JEV. CONCLUSIONS: Overall, this research shows that Swedish Cx. pipiens can become infected with JEV, and emphasizes the importance of further understanding of the thresholds and barriers for JEV dissemination in mosquitoes.

Implant Texture and Capsular Contracture: A Review of Cellular and Molecular Pathways.

Background: Capsular contracture (CC) is a leading cause of morbidity in implant-based breast surgery. Implant surface texture has been implicated in CC development, yet its etiopathogenesis remains unclear. We conducted a systematic review to determine the influence of implant surface texture on cellular and molecular mechanisms involved in the etiopathogenesis of CC. Methods: A systematic review of the MEDLINE, Embase, Web of Science, and Scopus databases was completed to examine the influence of implant texture on cellular and molecular pathways leading to CC. Excluded articles were reviews and those examining solely the clinical presentation of CC. Results: Development of CC includes prolonged inflammation, increased myofibroblast density, parallel arrangement of collagen fibers, and biofilm formation. When compared with textured implants, smooth implants are associated with reduction in parallel collagen, capsule thickness, and sheer frictional force. Microtextured implants trigger a reduced macrophage response and decreased fibroblast activation as compared with smooth and macrotextured surfaces. Bacterial counts on microtextured and smooth surfaces are significantly lower than that of macrotextured surfaces. Both micro- and macrotextured implants have increased matrix metalloproteinases and activation of tumor necrosis factor α pathway, with increased activation of the transforming growth factor ß1 pathway relative to smooth implants. Conclusions: Implant surface texture alters the cellular and molecular mechanisms in the chronic inflammatory process leading to CC. Given the complex biological system of cellular and molecular events in CC, a mathematical model integrating these influences may be optimal to deduce the etiopathogenesis.

The lysosomal trafficking regulator "LYST": an 80-year traffic jam.

Lysosomes and lysosome related organelles (LROs) are dynamic organelles at the intersection of various pathways involved in maintaining cellular hemostasis and regulating cellular functions. Vesicle trafficking of lysosomes and LROs are critical to maintain their functions. The lysosomal trafficking regulator (LYST) is an elusive protein important for the regulation of membrane dynamics and intracellular trafficking of lysosomes and LROs. Mutations to the LYST gene result in Chédiak-Higashi syndrome, an autosomal recessive immunodeficiency characterized by defective granule exocytosis, cytotoxicity, etc. Despite eight decades passing since its initial discovery, a comprehensive understanding of LYST's function in cellular biology remains unresolved. Accumulating evidence suggests that dysregulation of LYST function also manifests in other disease states. Here, we review the available literature to consolidate available scientific endeavors in relation to LYST and discuss its relevance for immunomodulatory therapies, regenerative medicine and cancer applications.

Genome resources for three modern cotton lines guide future breeding efforts.

Cotton (Gossypium hirsutum L.) is the key renewable fibre crop worldwide, yet its yield and fibre quality show high variability due to genotype-specific traits and complex interactions among cultivars, management practices and environmental factors. Modern breeding practices may limit future yield gains due to a narrow founding gene pool. Precision breeding and biotechnological approaches offer potential solutions, contingent on accurate cultivar-specific data. Here we address this need by generating high-quality reference genomes for three modern cotton cultivars ('UGA230', 'UA48' and 'CSX8308') and updating the 'TM-1' cotton genetic standard reference. Despite hypothesized genetic uniformity, considerable sequence and structural variation was observed among the four genomes, which overlap with ancient and ongoing genomic introgressions from 'Pima' cotton, gene regulatory mechanisms and phenotypic trait divergence. Differentially expressed genes across fibre development correlate with fibre production, potentially contributing to the distinctive fibre quality traits observed in modern cotton cultivars. These genomes and comparative analyses provide a valuable foundation for future genetic endeavours to enhance global cotton yield and sustainability.

Different memory systems in food-hoarding birds: A response to Pravosudov.

We recently showed that food-hoarding birds use familiarity processes more than recollection processes when remembering the spatial location of their caches (Smulders et al., Animal Cognition 26:1929-1943, 2023). Pravosudov (Learning & Behavior, https://doi.org/ https://doi.org/10.3758/s13420-023-00616-x , 2023) called our findings into question, claiming that our method is unable to distinguish between recollection and familiarity, and that associative learning tasks are a better way to study the memory for cache sites. In this response, we argue that our methods would have been more likely to detect recollection than familiarity, if Pravosudov's assertions were correct. We also point out that associative learning mechanisms may be good for building semantic knowledge, but are incompatible with the needs of cache site memory, which requires the unique encoding of caching events.

Expression in poplar of dehydroshikimate dehydratase induces transcriptional and metabolic changes in the phenylpropanoid pathway.

Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In-planta expression of a 3-dehydroshikimate dehydratase (QsuB) in poplar trees reduced lignin content and altered their monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we amassed fundamental knowledge on lignin-modified QsuB poplar using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibits the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. Changes affect predominantly the shikimate and phenylpropanoid pathways as wells as secondary cell wall metabolism, and result in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

Hippo and PI5P4K signaling intersect to control the transcriptional activation of YAP.

Phosphoinositides are essential signaling molecules. The PI5P4K family of phosphoinositide kinases and their substrates and products, PI5P and PI4,5P2, respectively, are emerging as intracellular metabolic and stress sensors. We performed an unbiased screen to investigate the signals that these kinases relay and the specific upstream regulators controlling this signaling node. We found that the core Hippo pathway kinases MST1/2 phosphorylated PI5P4Ks and inhibited their signaling in vitro and in cells. We further showed that PI5P4K activity regulated several Hippo- and YAP-related phenotypes, specifically decreasing the interaction between the key Hippo proteins MOB1 and LATS and stimulating the YAP-mediated genetic program governing epithelial-to-mesenchymal transition. Mechanistically, we showed that PI5P interacted with MOB1 and enhanced its interaction with LATS, thereby providing a signaling connection between the Hippo pathway and PI5P4Ks. These findings reveal how these two important evolutionarily conserved signaling pathways are integrated to regulate metazoan development and human disease.

Influence of Anatomic Locations of T1b Melanomas and Patient Age on Outcomes.

BACKGROUND: There are no guidelines on when to more strongly recommend sentinel lymph node biopsy (SLNB) for T1b melanomas. OBJECTIVE: To examine whether anatomic locations of T1b melanomas and patient age influence metastases. METHODS: We conducted a retrospective study using data from two hospitals in Los Angeles County from January 2010 through January 2020. RESULTS: Out of 620 patients with primary melanomas, 566 melanomas were staged based on the American Joint Committee on Cancer 8th edition melanoma staging. Forty-one were T1b, of which 13 were located on the face/ear/scalp and 28 were located elsewhere. T1b melanomas located on the face/ear/scalp had an increased risk of lymph node or distant metastasis compared with other anatomic sites (31% vs 3.6%, P=0.028). For all melanomas, the risk of lymph node or distant metastasis decreased with age of 64 years or greater (P<0.001 and P=0.034). For T1b melanomas, the risk of distant metastasis increased with increasing age (P=0.047). LIMITATIONS: Data were from a single county.  Conclusion: T1b melanomas of the face/ear/scalp demonstrated a higher risk of lymph node or distant metastasis and may help guide the recommendation of SLNB, imaging, and surveillance. Younger patients may be more strongly considered for SLNB and older patients with T1b melanomas may warrant imaging.  J Drugs Dermatol. 2024;23(5):306-310. doi:10.36849/JDD.7667.