The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunist bacterium that causes acute and chronic infections. During acute infections, the type III secretion system (T3SS) plays a pivotal role in allowing the bacteria to translocate effectors such as ExoS, ExoT, and ExoY into host cells for colonization. Previous research on the involvement of quorum sensing systems Las and Rhl in controlling the T3SS gene expression produced ambiguous results. In this study, we determined the role of the Las and Rhl systems and the PqsE protein on T3SS expression. Our results show that in the wild-type PAO1 strain, the deletion of lasR or pqsE do not affect the secretion of ExoS. However, rhlI inactivation increases the expression of T3SS genes. In contrast to the rhlI deletion, rhlR inactivation decreases both T3SS genes expression and ExoS secreted protein levels, and this phenotype is restored when this mutant is complemented with the exsA gene, which codes for the master regulator of the T3SS. Additionally, cytotoxicity is affected in the rhlR mutant strain compared with its PAO1 parental strain. Overall, our results indicate that neither the Las system nor PqsE are involved in regulating the T3SS. Moreover, the Rhl system components have opposite effects, RhlI participates in negatively controlling the T3SS expression, while RhlR does it in a positive way, and this regulation is independent of C4 or PqsE. Finally, we show that rhlR, rhlI, or pqsE inactivation abolished pyocyanin production in T3SS-induction conditions. The ability of RhlR to act as a positive T3SS regulator in the absence of its cognate autoinducer and PqsE shows that it is a versatile regulator that controls different virulence traits allowing P. aeruginosa to compete for a niche.

Learning from Tomorrow's Recyclers: Extension of Hands-on Recycled Waste Activity.

We previously developed a plastic sorting activity for high school students. In that activity, several tools were provided to separate plastic waste materials based on their physical properties while considering the time and cost for each separation step. Here, we aimed to understand the impact of this activity on the awareness of and learning about plastic sorting in a younger age group (8 to 11 years old) and explored how parental involvement influenced students' interest in the topic. The activity was part of the STEM Zone Saturday program at the University of Houston. Pre- and post-evaluations were used to assess students' understanding of plastic sorting and overall experiences. While some insights into plastic sorting were derived from previous experiences, the activity improved awareness among students, particularly regarding sorting based on physical properties. The involvement of parents encouraged exploration, discovery, and enjoyment. These findings underscore the importance of early education and community engagement in fostering sustainable practices and advancing toward a circular economy.

Encorafenib and binimetinib followed by radiotherapy for patients with BRAFV600-mutant melanoma and brain metastases (E-BRAIN/GEM1802 phase II study).

BACKGROUND: Encorafenib plus binimetinib (EB) is a standard of care treatment for advanced BRAFV600-mutant melanoma. We assessed efficacy and safety of encorafenib plus binimetinib in patients with BRAFV600-mutant melanoma and brain metastasis (BM) and explored if radiotherapy improves the duration of response. METHODS: E-BRAIN/GEM1802 was a prospective, multicenter, single arm, phase II trial that enrolled patients with melanoma BRAFV600-mutant and BM. Patients received encorafenib 450 mg once daily plus binimetinib 45 mg BID, and those who achieved partial response or stable disease at first tumor assessment were offered radiotherapy. Treatment continued until progression.Primary endpoint was intracranial response rate (icRR) after 2 months of EB, establishing a futility threshold of 60%. RESULTS: The study included 25 patients with no BM symptoms and 23 patients with BM symptoms regardless of using corticosteroids. Among them, 31 patients (64.6%) received sequential radiotherapy. After two months, icRR was 70.8% (95% CI: 55.9-83.1); 10.4% complete response. Median intracranial PFS and OS were 8.5 (95% CI: 6.4-11.8) and 15.9 (95% CI: 10.7-21.4) months, respectively (8.3 months for icPFS and 13.9 months OS for patients receiving RDT). Most common grade 3-4 treatment-related adverse event was alanine aminotransferase (ALT) increased (10.4%). CONCLUSION: Encorafenib plus binimetinib showed promising clinical benefit in terms of icRR, and tolerable safety profile with low frequency of high grade TRAEs, in patients with BRAFV600-mutant melanoma and BM, including those with symptoms and need for steroids. Sequential radiotherapy is feasible but it does not seem to prolong response.

Access to systemic treatment of non-melanoma skin cancer in Spain: a survey analysis.

BACKGROUND: Novel and highly effective drugs for non-melanoma skin cancer (NMSC) improve patient outcomes, but their high cost strains healthcare systems. Spain's decentralized public health system, managed by 17 autonomous communities (AaCc), raises concerns about equitable access. METHODS: A cross-sectional survey (July-September 2023) was sent to Spanish Multidisciplinary Melanoma Group (GEM Group) members to assess access to new drugs. FINDINGS: Fifty physicians from 15 Spanish AaCc responded to the survey. Access for drug with approved public reimbursement, Hedgehog inhibitors in basal-cell carcinoma and anti PD-L1 antibody in Merkel carcinoma, was observed in 84% and 86% of centers, respectively. For other EMA-approved treatments, but without reimbursement in Spain access decreased to 78% of centers. Heterogeneity in access was mainly observed intra regions. CONCLUSION: Unequal financial support for drugs for NMSC with creates a patchwork of access across Spanish hospitals, with variations even within the same AaCc.

Antivirulence and antipathogenic activity of Mayan herbal remedies against Pseudomonas aeruginosa.

ETHNOPHARMACOLOGICAL RELEVANCE: The Yucatan Peninsula has a privileged wealth of vascular plants with which various Mayan herbal formulations have been developed. However, studies on their antipathogenic and antivirulence properties are scarce. AIM OF THE STUDY: Identify antivirulence properties in Mayan herbal remedies and determine their antipathogenic capacity in burn wounds infected with Pseudomonas aeruginosa. MATERIALS AND METHODS: An ethnobotanical study was conducted in Mayan communities in central and southern Quintana Roo, Mexico. Furthermore, the antipathogenic capacity of three Mayan herbal remedies was analyzed using an animal model of thermal damage and P. aeruginosa infection. Antivirulence properties were determined by inhibiting phenotypes regulated by quorum sensing (pyocyanin, biofilm, and swarming) and by the secretion of the ExoU toxin. The chemical composition of the most active herbal remedy was analyzed using molecular network analysis. RESULTS: It was found that topical administration of the remedy called "herbal soap" (HS) for eleven days maintained 100% survival of the animals, reduced establishment of the bacteria in the burn and prevented its systemic dispersion. Although no curative effect was recorded on tissue damaged by HS treatment, its herbal composition strongly reduced swarming and ExoU secretion. Through analysis of Molecular Networks, it was possible to carry out a global study of its chemical components, and identify the family of oxindole monoterpenoid alkaloids and carboline and tetrahydropyrididole alkaloids. In addition, flavonols, flavan-3-ols, and quinic acid derivatives were detected. CONCLUSIONS: The antipathogenic and antivirulence capacity of ancient Mayan remedies makes them a potential resource for developing new antibacterial therapies to treat burns infected by P. aeruginosa.

Access to melanoma drugs in Spain: a cross-sectional survey.

BACKGROUND: The development of highly active drugs has improved the survival of melanoma patients, but elevated drug prices place a significant burden on health care systems. In Spain, the public health care system is transferred to the 17 autonomous communities (AACC). The objective of this study is to describe the situation of drug access for melanoma patients in Spain and how this decentralized system is affecting equity. METHODS: From July to September 2023, a cross-sectional survey was sent to members of the Spanish Multidisciplinary Melanoma Group (GEM Group). The questionnaire consulted about the real access to new drugs in each hospital. The responses were collected anonymously and analyzed according to several variables, including the AACC. RESULTS: The survey was answered by 50 physicians in 15 AACC. No major differences on access between AACC were observed for indications that are reimbursed by the Spanish Health Care System (adjuvant immunotherapy for stage IIIC-IIID and resected stage IV melanoma). Important differences in drug access were observed among AACC and among centers within the same AACC, for most of the EMA indications that are not reimbursed (adjuvant immunotherapy for stages IIB-IIC-IIIA-IIIB) or that are not fully reimbursed (ipilimumab plus nivolumab in advanced stage). Homogeneously, access to adjuvant targeted drugs, TIL therapy and T-VEC, is extremely low or non-existing in all AACC. CONCLUSIONS: For most indications that reimbursement is restricted out of the EMA indication, a great diversity on access was found throughout the different hospitals in Spain, including heterogeneity intra-AACC.

SEOM-GEM clinical guidelines for cutaneous melanoma (2023).

Cutaneous melanoma incidence is rising. Early diagnosis and treatment administration are key for increasing the chances of survival. For patients with locoregional advanced melanoma that can be treated with complete resection, adjuvant-and more recently neoadjuvant-with targeted therapy-BRAF and MEK inhibitors-and immunotherapy-anti-PD-1-based therapies-offer opportunities to reduce the risk of relapse and distant metastases. For patients with advanced disease not amenable to radical treatment, these treatments offer an unprecedented increase in overall survival. A group of medical oncologists from the Spanish Society of Medical Oncology (SEOM) and Spanish Multidisciplinary Melanoma Group (GEM) has designed these guidelines, based on a thorough review of the best evidence available. The following guidelines try to cover all the aspects from the diagnosis-clinical, pathological, and molecular-staging, risk stratification, adjuvant therapy, advanced disease therapy, and survivor follow-up, including special situations, such as brain metastases, refractory disease, and treatment sequencing. We aim help clinicians in the decision-making process.

Análisis retrospectivo de las hipoglucemias inducidas por venlafaxina en pacientes con sobredosis / Retrospective analysis of venlafaxine-induced hypoglycemia in patients with overdose

Introducción: Estudios recientes en intoxicaciones por venlafaxina (VLF) relacionan la presencia de hipoglucemia con la dosis. Nuestro objetivo fue analizar las características clínicas de los pacientes que presentaron hipoglucemia inducida por sobredosis de VLF.Pacientes y métodosEstudio retrospectivo realizado en las Islas Baleares (2020-2023). Como criterios de inclusión se tomaron en cuenta las concentraciones séricas de VLF + ortodesmetilvenlafaxina (O-VLF) > 800 ng/mL. Se compararon las características de los pacientes con y sin hipoglucemia.ResultadosSe incluyeron 21 pacientes, ocho (38,1%) con hipoglucemia. No se hallaron diferencias en las dosis referidas en ambos grupos. Las concentraciones máximas de VLF + O-VLF (ng/mL) fueron 9.783 (4.459-17.976) en sujetos con hipoglucemia y 1.413 (930-1.769) en aquellos sin esta enfermedad (p<0,0001). La presencia de hipoglucemia se asoció con: menor edad y nivel de conciencia; y mayor frecuencia de tentativas suicidas, convulsiones, midriasis, taquicardia y síndrome serotoninérgico, soporte respiratorio invasivo, sueroterapia e ingreso en la Unidad de Cuidados Intensivos (UCI) (p < 0,05).ConclusionesLa detección de hipoglucemia en individuos intoxicados por VLF es un marcador fácilmente disponible para sospechar la gravedad del paciente. En cualquier caso, las concentraciones séricas, cuando se disponen, permiten confirmar la intoxicación. (AU)

Introduction: Recent publications relate the presence of hypoglycemia in venlafaxine (VLX) poisoning depending on the dose. Our aim was to analyze the clinical characteristics of patients who presented hypoglycemia induced by VLF overdose.Patients and methodsRetrospective study carried out in the Balearic Islands (2020–2023). Inclusion criteria: serum concentrations of VLX + O-desmethyl-venlafaxine (O-VLX)>800 ng/mL. The characteristics of patients with and without hypoglycemia were compared.ResultsTwenty-one patients were included, 8 (38.1%) with hypoglycemia. No differences were found in the doses referred to in both groups. Peak concentrations of VLX + O-VLX (ng/mL) were 9,783 [4,459–17,976] in patients with hypoglycemia and 1,413 [930–1,719] in patients without hypoglycemia (p<0.0001). The presence of hypoglycemia was associated with: lower age and level of consciousness; and higher frequency of suicide attempts, seizures, mydriasis, tachycardia and serotonin syndrome, invasive respiratory support, fluid therapy and ICU admission (p<0.05).ConclusionsThe detection of hypoglycemia in a VLX overdose case is a readily available marker to suspect the severity of the patient. In any case, serum concentrations when available allow us to confirm intoxication. (AU)

[Retrospective analysis of venlafaxine-induced hypoglycemia in patients with overdose]. / Análisis retrospectivo de las hipoglucemias inducidas por venlafaxina en pacientes con sobredosis.

INTRODUCTION: Recent publications relate the presence of hypoglycemia in venlafaxine (VLX) poisoning depending on the dose. Our aim was to analyze the clinical characteristics of patients who presented hypoglycemia induced by VLF overdose. PATIENTS AND METHODS: Retrospective study carried out in the Balearic Islands (2020-2023). INCLUSION CRITERIA: serum concentrations of VLX + O-desmethyl-venlafaxine (O-VLX)>800 ng/mL. The characteristics of patients with and without hypoglycemia were compared. RESULTS: Twenty-one patients were included, 8 (38.1%) with hypoglycemia. No differences were found in the doses referred to in both groups. Peak concentrations of VLX + O-VLX (ng/mL) were 9,783 [4,459-17,976] in patients with hypoglycemia and 1,413 [930-1,719] in patients without hypoglycemia (p<0.0001). The presence of hypoglycemia was associated with: lower age and level of consciousness; and higher frequency of suicide attempts, seizures, mydriasis, tachycardia and serotonin syndrome, invasive respiratory support, fluid therapy and ICU admission (p<0.05). CONCLUSIONS: The detection of hypoglycemia in a VLX overdose case is a readily available marker to suspect the severity of the patient. In any case, serum concentrations when available allow us to confirm intoxication.

Opposing Effects on Descending Control of Nociception by µ and κ Opioid Receptors in the Anterior Cingulate Cortex.

BACKGROUND: The efficiency of descending pain modulation, commonly assessed with the conditioned pain modulation procedure, is diminished in patients with chronic pain. The authors hypothesized that the efficiency of pain modulation is controlled by cortical opioid circuits. METHODS: This study evaluated the effects of µ opioid receptor activation in the anterior cingulate cortex on descending control of nociception, a preclinical correlate of conditioned pain modulation, in male Sprague-Dawley rats with spinal nerve ligation-induced chronic pain or in sham-operated controls. Additionally, the study explored the consequences of respective activation or inhibition of κ opioid receptor in the anterior cingulate cortex of naive rats or animals with neuropathic pain. Descending control of nociception was measured as the hind paw withdrawal response to noxious pressure (test stimulus) in the absence or presence of capsaicin injection in the forepaw (conditioning stimulus). RESULTS: Descending control of nociception was diminished in the ipsilateral, but not contralateral, hind paw of rats with spinal nerve ligation. Bilateral administration of morphine in the anterior cingulate cortex had no effect in shams but restored diminished descending control of nociception without altering hypersensitivity in rats with neuropathic pain. Bilateral anterior cingulate cortex microinjection of κ opioid receptor antagonists, including nor-binaltorphimine and navacaprant, also re-established descending control of nociception in rats with neuropathic pain without altering hypersensitivity and with no effect in shams. Conversely, bilateral injection of a κ opioid receptor agonist, U69,593, in the anterior cingulate cortex of naive rats inhibited descending control of nociception without altering withdrawal thresholds. CONCLUSIONS: Anterior cingulate cortex κ opioid receptor activation therefore diminishes descending control of nociception both in naive animals and as an adaptive response to chronic pain, likely by enhancing net descending facilitation. Descending control of nociception can be restored by activation of µ opioid receptors in the anterior cingulate cortex, but also by κ opioid receptor antagonists, providing a nonaddictive alternative to opioid analgesics. Navacaprant is now in advanced clinical trials.

A Phase II Trial of the CD40 Agonistic Antibody Sotigalimab (APX005M) in Combination with Nivolumab in Subjects with Metastatic Melanoma with Confirmed Disease Progression on Anti-PD-1 Therapy.

PURPOSE: Disease progression during or after anti-PD-1-based treatment is common in advanced melanoma. Sotigalimab is a CD40 agonist antibody with a unique epitope specificity and Fc receptor binding profile optimized for activation of CD40-expressing antigen-presenting cells. Preclinical data indicated that CD40 agonists combined with anti-PD1 could overcome resistance to anti-PD-1. PATIENTS AND METHODS: We conducted a multicenter, open-label, phase II trial to evaluate the combination of sotigalimab 0.3 mg/kg and nivolumab 360 mg every 3 weeks in patients with advanced melanoma following confirmed disease progression on a PD-1 inhibitor. The primary objective was to determine the objective response rate (ORR). RESULTS: Thirty-eight subjects were enrolled and evaluable for safety. Thirty-three were evaluable for activity. Five confirmed partial responses (PR) were observed for an ORR of 15%. Two PRs are ongoing at 45.9+ and 26+ months, whereas the other three responders relapsed at 41.1, 18.7, and 18.4 months. The median duration of response was at least 26 months. Two additional patients had stable disease for >6 months. Thirty-four patients (89%) experienced at least one adverse event (AE), and 13% experienced a grade 3 AE related to sotigalimab. The most common AEs were pyrexia, chills, nausea, fatigue, pruritus, elevated liver function, rash, vomiting, headache, arthralgia, asthenia, myalgia, and diarrhea. There were no treatment-related SAEs, deaths, or discontinuation of sotigalimab due to AEs. CONCLUSIONS: Sotigalimab plus nivolumab had a favorable safety profile consistent with the toxicity profiles of each agent. The combination resulted in durable and prolonged responses in a subset of patients with anti-PD-1-resistant melanoma, warranting further evaluation in this setting. See related commentary by Wu and Luke, p. 9.

Efficacy and safety of cosibelimab, an anti-PD-L1 antibody, in metastatic cutaneous squamous cell carcinoma.

BACKGROUND: Programmed cell death receptor-1 (PD-1)-blocking antibodies are approved to treat metastatic or locally advanced cutaneous squamous cell carcinoma (CSCC) cases ineligible for curative surgery or radiation. Notwithstanding, some patients experience inadequate responses or severe immune-related adverse events (AEs), indicating the need for improved therapies. Cosibelimab is a high-affinity programmed cell death-ligand 1 (PD-L1)-blocking antibody that activates innate and adaptive immunity by blocking PD-L1 interaction with PD-1 and B7-1 receptors. It is an unmodified immunoglobulin G1 subtype with a functional Fc domain capable of inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Here, we present results of the pivotal study of patients with metastatic CSCC from an open-label, multicenter, multiregional, multicohort, phase 1 trial of cosibelimab. METHODS: In this trial, participants with metastatic CSCC received cosibelimab 800 mg intravenously every 2 weeks. Primary endpoint was objective response rate (ORR) by independent central review using Response Evaluation Criteria in Solid Tumors, V.1.1. Secondary endpoints included duration of response (DOR) and safety. RESULTS: Objective response was observed in 37 of 78 participants (47.4% (95% CI: 36.0% to 59.1%)), with median follow-up of 15.4 months (range: 0.4 to 40.5) as of data cut-off. Median DOR was not reached (range: 1.4+ to 34.1+ months), with response ongoing in 73.0% of participants. Common treatment-emergent AEs (≥15%) were fatigue (26.9%), rash (16.7%), and anemia (15.4%). Eighteen participants (23.1%) experienced immune-related AEs (grade 3: n=2 (2.6%); no grade 4/5). No treatment-related deaths were reported. CONCLUSIONS: Cosibelimab demonstrated clinically meaningful ORR and DOR and was associated with a manageable safety profile. TRIAL REGISTRATION NUMBER: NCT03212404.

To cheat or not to cheat: cheatable and non-cheatable virulence factors in Pseudomonas aeruginosa.

Important bacterial pathogens such as Pseudomonas aeruginosa produce several exoproducts such as siderophores, degradative enzymes, biosurfactants, and exopolysaccharides that are used extracellularly, benefiting all members of the population, hence being public goods. Since the production of public goods is a cooperative trait, it is in principle susceptible to cheating by individuals in the population who do not invest in their production, but use their benefits, hence increasing their fitness at the expense of the cooperators' fitness. Among the most studied virulence factors susceptible to cheating are siderophores and exoproteases, with several studies in vitro and some in animal infection models. In addition to these two well-known examples, cheating with other virulence factors such as exopolysaccharides, biosurfactants, eDNA production, secretion systems, and biofilm formation has also been studied. In this review, we discuss the evidence of the susceptibility of each of those virulence factors to cheating, as well as the mechanisms that counteract this behavior and the possible consequences for bacterial virulence.

First Trimester Evaluation of Maternal Visceral Fat and Its Relationship with Adverse Pregnancy Outcomes.

Obese women are more likely to experience pregnancy complications. The distribution of fat, and more particularly the rise in visceral fat, is well established to be more closely linked to the onset of cardiovascular disease and metabolic syndrome than obesity itself. We aim to examine the relationship between maternal visceral fat assessment in the first trimester and the appearance of adverse pregnancy outcomes. A prospective cohort study including 416 pregnant women was conducted. During the first trimester scan (11-13 + 6 weeks), all individuals had their visceral fat and subcutaneous thicknesses measured by ultrasonography. Blood samples were obtained, and maternal demographics and clinical information were documented. After delivery, the obstetric outcomes were evaluated. We contrasted two groups: one with healthy pregnancies and the other with adverse pregnancy outcomes (APO), defined as the development of at least one of the following complications: gestational diabetes mellitus, hypertensive disorders of pregnancy, abnormal fetal growth, preterm delivery or preterm premature rupture of membranes. Median maternal age was 33 and 34 years old for the uncomplicated and adverse pregnancy outcomes groups, respectively. We found that women with adverse pregnancy outcomes had higher VFT (median 30 vs. 26.5 mm, p = 0.001) and SFT (median 18.9 vs. 17.1 mm, p = 0.03). However, the visceral/subcutaneous fat ratio was not statistically different between groups. Finally, we performed a subanalysis for metabolic and placental vascular dysfunction complications. After performing a multivariate logistic regression analysis adjusted for maternal age, smoking, and mean arterial pressure, both the VFT (aOR 1.03, p < 0.001) and the ratio of visceral/subcutaneous fat (aOR 1.37, p = 0.04) were significantly associated with the development of adverse pregnancy outcomes; however, the associations of VFT and the VFT-to-SFT ratio were higher for the occurrence of gestational diabetes (aOR 1.07, p < 0.001; aOR 2.09, p = 0.001; respectively) and showed no relationships with placental complications. When conducting a first-trimester ultrasound assessment, sonographers may measure VFT without additional time or cost involved. Identification of pregnant women with increased VFT (>37 mm) may benefit from a close follow-up, especially for the development of gestational diabetes, independent of BMI.

Corneal topography and tomography readings with mask wear during the COVID-19 pandemic.

PURPOSE: To assess the effect of mask wear on corneal topography and tomography readings. SETTING: Tertiary ophthalmic center. DESIGN: Prospective, observational, comparative study. METHODS: Subjects underwent imaging with the Tomey TMS-4a topographer and the Oculus Pentacam HR tomographer. Imaging was performed without a mask, and then testing was repeated while wearing 3 different masks: Halyard Level 2, Halyard Level 3, and KN95. Measurements during mask wear were compared with measurements without a mask. The Kruskal-Wallis analysis of variance grouped test was used to compare mean differences without vs with mask wear on measurements. The Chi-square test was used to compare frequency of differences between different masks and against-the-rule vs with-the-rule astigmatism. The frequencies of clinically significant changes in axis of astigmatism, magnitude of astigmatism, and mean keratometry (K) values were calculated. RESULTS: 52 eyes were included in the study with a mean age of 34.71. Mask wear did not show statistically significant differences in mean topography and tomography measurements compared with without mask wear for all parameters. However, the majority, 53% (83/156) had a >10 degrees and 41% (64/156) had >15 degrees change in axis of astigmatism on topography when wearing a mask compared with no mask wear. CONCLUSIONS: Mask wear did not result in statistically significant mean changes in K readings on topography and tomography. However, the axis of astigmatism varied >10 degrees in most of the patients. Axis determination should be interpreted with caution regarding refractive surgery and/or toric intraocular lens planning in individuals whose measurements were obtained while wearing a mask.

Chronic pain recruits hypothalamic dynorphin/kappa opioid receptor signalling to promote wakefulness and vigilance.

Increased vigilance in settings of potential threats or in states of vulnerability related to pain is important for survival. Pain disrupts sleep and conversely, sleep disruption enhances pain, but the underlying mechanisms remain unknown. Chronic pain engages brain stress circuits and increases secretion of dynorphin, an endogenous ligand of the kappa opioid receptor (KOR). We therefore hypothesized that hypothalamic dynorphin/KOR signalling may be a previously unknown mechanism that is recruited in pathological conditions requiring increased vigilance. We investigated the role of KOR in wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in freely moving naïve mice and in mice with neuropathic pain induced by partial sciatic nerve ligation using EEG/EMG recordings. Systemic continuous administration of U69,593, a KOR agonist, over 5 days through an osmotic minipump decreased the amount of NREM and REM sleep and increased sleep fragmentation in naïve mice throughout the light-dark sleep cycle. We used KORcre mice to selectively express a Gi-coupled designer receptor activated by designer drugs (Gi-DREADD) in KORcre neurons of the hypothalamic paraventricular nucleus, a key node of the hypothalamic-pituitary-adrenal stress response. Sustained activation of Gi-DREADD with clozapine-N-oxide delivered in drinking water over 4 days, disrupted sleep in these mice in a similar way as systemic U69,593. Mice with chronic neuropathic pain also showed disrupted NREM and total sleep that was normalized by systemic administration of two structurally different KOR antagonists, norbinaltorphimine and NMRA-140, currently in phase II clinical development, or by CRISPR/Cas9 editing of paraventricular nucleus KOR, consistent with endogenous KOR activation disrupting sleep in chronic pain. Unexpectedly, REM sleep was diminished by either systemic KOR antagonist or by CRISPR/Cas9 editing of paraventricular nucleus KOR in sham-operated mice. Our findings reveal previously unknown physiological and pathophysiological roles of dynorphin/KOR in eliciting arousal. Physiologically, dynorphin/KOR signalling affects transitions between sleep stages that promote REM sleep. Furthermore, while KOR antagonists do not promote somnolence in the absence of pain, they normalized disrupted sleep in chronic pain, revealing a pathophysiological role of KOR signalling that is selectively recruited to promote vigilance, increasing chances of survival. Notably, while this mechanism is likely beneficial in the short-term, disruption of the homeostatic need for sleep over longer periods may become maladaptive resulting in sustained pain chronicity. A novel approach for treatment of chronic pain may thus result from normalization of chronic pain-related sleep disruption by KOR antagonism.

Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa.

Background: Bacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance. Methods: In this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains. Results: Phage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia. Conclusions: We show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy's evolutionary impact that may be exploited to generate robust therapy schemes.

Fetal surgery for occipital encephalocele: A case report.

Background: Occipital encephalocele is a congenital defect of the neural tube at the level of the cranial midline, which results in herniation of meninges and brain tissue. The results of the management of myelomeningocele study determine the maternal and fetal risks for an open fetal surgery and have motivated the constant review of the concepts and strategies which the pediatric neurosurgeon can employ for the treatment of neural tube defects in the prenatal period. Case Description: We present a case of a female patient in utero of 26 gestational weeks with the diagnosis of an occipital encephalocele treated by open fetal surgery. During week 20 of gestation, the diagnosis of occipital encephalocele was made by ultrasound, which was corroborated by fetal magnetic resonance that showed cranial protrusion of neural and meningeal content in the occipital region, measuring 1.6 × 2.8 × 3.3 cm with an approximate volume of 7.7 cc through a bone defect of 6 mm. The closure of the defect was performed by the postnatal surgical technique adapted to the open fetal surgery. Later, the patient was born transabdominal with a 2.8 cm occipital wound, with suture points and approximated borders, normocephalic, without clinical signs of sepsis, hydrocephalus, or overt neurologic compromise. Conclusion: Open fetal surgery is a therapeutic option in the face of an isolated occipital encephalocele. This case report demonstrates the viability of the surgical procedure by the adaptation of a postnatal surgical technique to a prenatal surgery. Further studies are needed to evaluate the long-term functional results, comparing them with those seen in patients who undergo a postnatal procedure.

Association of Circular RNA and Long Non-Coding RNA Dysregulation with the Clinical Response to Immune Checkpoint Blockade in Cutaneous Metastatic Melanoma.

Cutaneous melanoma (CM) is the most lethal form of skin cancer if it becomes metastatic, where treatment options and survival chances decrease dramatically. Immunotherapy treatments based on the immunologic checkpoint inhibitors programmed death cell protein 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) constituted a main breakthrough in the treatment of metastatic CM, particularly for the achievement of long-term benefits. Even though it is a very promising therapy, resistance to primary immune checkpoint blockade (ICB) arises in about 70% of CM patients treated with a CTLA-4 inhibitor, and 40-65% of CM patients administered with a PD-1-targeting treatment. Some long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are implicated in triggering pro- and anti-tumorigenic responses to various cancer treatments. The relationship between lncRNAs, circRNAs and ICB immunotherapy has not been explored in cutaneous metastatic melanoma (CMM). The aim of this pilot study is to evaluate the potential role of circRNA and lncRNA expression variability as pre-treatment predictor of the clinical response to immunotherapy in CMM patients. RNA-seq from 12 formalin-fixed paraffin-embedded (FFPE) samples from the metastatic biopsies of CMM patients treated with nivolumab was used to identify response-associated transcripts. Our findings indicate that specific lncRNAs and circRNAs, probably acting as competitive endogenous RNAs (ceRNAs), are involved in the regulatory networks of the immune response against metastatic melanoma that these patients have under treatment with nivolumab. Moreover, we established a risk score that yields predictions of the overall survival (OS) and progression-free survival (PFS) of CMM patients with high accuracy. This proof-of-principle work provides a possible insight into the function of ceRNAs, contributing to efforts to decipher the complex molecular mechanisms of ICB cancer treatment response.

Gut Microbiota and Therapy in Metastatic Melanoma: Focus on MAPK Pathway Inhibition.

Gut microbiome (GM) and its either pro-tumorigenic or anti-tumorigenic role is intriguing and constitutes an evolving landscape in translational oncology. It has been suggested that these microorganisms may be involved in carcinogenesis, cancer treatment response and resistance, as well as predisposition to adverse effects. In melanoma patients, one of the most immunogenic cancers, immune checkpoint inhibitors (ICI) and MAPK-targeted therapy-BRAF/MEK inhibitors-have revolutionized prognosis, and the study of the microbiome as a modulating factor is thus appealing. Although BRAF/MEK inhibitors constitute one of the main backbones of treatment in melanoma, little is known about their impact on GM and how this might correlate with immune re-induction. On the contrary, ICI and their relationship to GM has become an interesting field of research due to the already-known impact of immunotherapy in modulating the immune system. Immune reprogramming in the tumor microenvironment has been established as one of the main targets of microbiome, since it can induce immunosuppressive phenotypes, promote inflammatory responses or conduct anti-tumor responses. As a result, ongoing clinical trials are evaluating the role of fecal microbiota transplant (FMT), as well as the impact of using dietary supplements, antibiotics and probiotics in the prediction of response to therapy. In this review, we provide an overview of GM's link to cancer, its relationship with the immune system and how this may impact response to treatments in melanoma patients. We also discuss insights about novel therapeutic approaches including FMT, changes in diet and use of probiotics, prebiotics and symbiotics. Finally, we hypothesize on the possible pathways through which GM may impact anti-tumor efficacy in melanoma patients treated with targeted therapy, an appealing subject of which little is known.