Deciphering the molecular components of the quorum sensing system in the fungus Ophiostoma piceae.

IMPORTANCE: This manuscript presents a comprehensive study on the molecular mechanisms triggered by the quorum sensing (QS) molecule farnesol in the biotechnologically relevant fungus Ophiostoma piceae. We present for the first time, using a multiomics approach, an in-depth analysis of the QS response in a saprotroph fungus, detailing the molecular components involved in the response and their possible mechanisms of action. We think that these results are particularly relevant in the knowledge of the functioning of the QS in eukaryotes, as well as for the exploitation of these mechanisms.

Substance Use Experiences Among Bisexual, Pansexual, and Queer (Bi+) Male Youth: A Qualitative Study of Motivations, Consequences, and Decision Making.

Sexual minority youth are at increased risk of substance use compared to their heterosexual peers, and bisexual youth appear to be at greatest risk. However, little is known about their motivations for and against using substances, how they make decisions, and what consequences they experience. We used qualitative data from a study of 54 cisgender and transgender male youth (ages 14-17 years) who reported attractions to more than one gender or regardless of gender (i.e., bisexual, pansexual, or queer; collectively referred to as bi+) to explore these aspects of substance use. Participants completed a survey and an interview, and interviews were thematically analyzed. Qualitative analyses revealed that participants described diverse motivations for using substances (e.g., to cope with stress, to experiment, to have fun) and for not using them (e.g., concern about consequences, not having access). The most common sources of stress were mental health problems, school, and family. They did not describe sexual orientation-related stress as a motivation for their use, but they acknowledged that it could influence others' use. Participants also described thinking about when, where, and with whom they were going to use prior to doing so (e.g., only using in safe places and with people who they trusted). Finally, they described a range of consequences they experienced (e.g., getting sick, getting in trouble), and a subset of transgender participants described experiencing dependence symptoms. These findings suggest that substance use prevention and harm reduction interventions for bi+ male youth should address diverse motivations for use, including general stressors, which are often overlooked compared to minority-specific stressors. Further, interventions should approach youth as capable of making decisions. Findings also highlight the particular need to address substance use among transgender youth.

A mixed methods study of sexuality education experiences and preferences among bisexual, pansexual, and queer (bi+) male youth.

Introduction: Bisexual male youth are more likely to engage in certain behaviors that contribute to HIV/STI transmission (e.g., substance use) than are heterosexual and gay male youth. However, sexuality education rarely addresses the unique needs of sexual minority youth, especially bisexual, pansexual, and queer (bi+) youth, and little is known about their sexuality education experiences and preferences. As such, the goal of this study was to examine bi+ male youth's experiences learning about sex and their preferences for sexuality education. Methods: In 2019, 56 bi+ male youth ages 14-17 were surveyed and interviewed about their sexuality education experiences and preferences. Participants identified as bisexual (64%), pansexual (27%), and queer (9%), were racially/ethnically diverse (39% white, 32% Latinx, 20% Black, 9% other races), and included cisgender (79%) and transgender (21%) male youth. Results: Participants described varied experiences with school-based sexuality education (e.g., none, abstinence only, covered sexual health in some way), but it rarely addressed their unique needs. They typically learned about sex by searching for information online and from sexually explicit media. Participants identified several topics they wanted to learn more about (e.g., sex with same-gender partners, anal sex, consent), but they typically believed they were prepared to have sex. Finally, some participants described benefits of tailoring sexuality education to their unique needs, while others described benefits of more inclusive programs. Conclusions and Policy Implications: Findings suggest that bi+ male youth do not receive adequate sexuality education to make informed decisions about safer sex, highlighting the critical need for reform.

Is hysterosalpingo-foam sonography (HyFoSy) more tolerable in terms of pain and anxiety than hysterosalpingography (HSG)? A prospective real-world setting multicentre study.

BACKGROUND: In 60% of sterile couples a female factor is present, with these being tubal factors in 30-50% of cases. A tubal patency test is also required in women without a male partner undergoing fertility treatment. Thus, an accurate, safe and tolerable technique should be available. The aim of this study is to determine and to compare hysterosalpingo-foam sonography (HyFoSy) and hysterosalpingography (HSG) tolerability in terms of pain and anxiety. METHODS: This is a prospective real-world setting multicentre study conducted in two tertiary hospitals in Madrid. 210 infertile women/women without a male partner looking to get pregnant were recruited; 111 for the HyFoSy group and 99 for the HSG group. Tolerability was measured in terms of anxiety by the State Trait Anxiety Inventory (STAI) and pain by the Visual Analogue Scale (VAS). RESULTS: Median VAS score in HyFoSy group was 2 (P25; P75: 1; 3) versus 5 (4; 8) in HSG group, p < 0.001. The median State-STAI score in the HSG group was 18 points (10; 26) versus 10 (7; 16) in the HyFoSy group (p < 0.001); the median Trait-STAI score in the HSG group was 15 (11; 21) versus 13 (9; 17) in the HyFoSy group (p = 0.044). CONCLUSIONS: HyFoSy shows higher tolerability to both: pain and anxiety. It is related to less pain and less post-test anxiety than HSG.

CommunityRx: Optimizing a Community Resource Referral Intervention for Minority Dementia Caregivers.

Community resource referral systems have been implemented into care settings that serve persons with dementia but with little input from caregivers. Focus groups were conducted with African American, Hispanic, and Asian caregivers to describe their preferences for community resource referral information. Caregivers discussed the significance of a community resource list for dementia caregiving and self-care and articulated strategies for effective information delivery during a medical visit. Most caregivers acknowledged that resource needs change with progression of dementia, but no patterns emerged with regard to preference for information delivered incrementally based on disease stage or all at once. Hispanic and Asian caregivers felt that resource information should specify service providers' language and cultural capabilities. All caregivers agreed that delivery by a member of the care team with knowledge of dementia-specific resources would be most effective. Optimal delivery of community resource referrals is caregiver-centered and customizable to individual and subgroup preferences.

Blood-brain barrier opening with focused ultrasound in Parkinson's disease dementia.

MR-guided focused ultrasound (MRgFUS), in combination with intravenous microbubble administration, has been applied for focal temporary BBB opening in patients with neurodegenerative disorders and brain tumors. MRgFUS could become a therapeutic tool for drug delivery of putative neurorestorative therapies. Treatment for Parkinson's disease with dementia (PDD) is an important unmet need. We initiated a prospective, single-arm, non-randomized, proof-of-concept, safety and feasibility phase I clinical trial (NCT03608553), which is still in progress. The primary outcomes of the study were to demonstrate the safety, feasibility and reversibility of BBB disruption in PDD, targeting the right parieto-occipito-temporal cortex where cortical pathology is foremost in this clinical state. Changes in ß-amyloid burden, brain metabolism after treatments and neuropsychological assessments, were analyzed as exploratory measurements. Five patients were recruited from October 2018 until May 2019, and received two treatment sessions separated by 2-3 weeks. The results are set out in a descriptive manner. Overall, this procedure was feasible and reversible with no serious clinical or radiological side effects. We report BBB opening in the parieto-occipito-temporal junction in 8/10 treatments in 5 patients as demonstrated by gadolinium enhancement. In all cases the procedures were uneventful and no side effects were encountered associated with BBB opening. From pre- to post-treatment, mild cognitive improvement was observed, and no major changes were detected in amyloid or fluorodeoxyglucose PET. MRgFUS-BBB opening in PDD is thus safe, reversible, and can be performed repeatedly. This study provides encouragement for the concept of BBB opening for drug delivery to treat dementia in PD and other neurodegenerative disorders.

Laparoscopic Partial Splenectomy Assisted by Fluorescence in a 13-Year-Old Girl.

Partial splenectomy allows preserving immune function in benign splenic lesions such as epidermoid cysts. Determining the plane of resection and perfusion of the spleen remnant can be difficult, especially in centrally located lesions. We present a 13-year-old girl with a symptomatic splenic cyst of 6 cm in diameter located next to the splenic hilum. Laparoscopic partial splenectomy was performed through a 10-mm umbilical approach and three accessory 5-mm ports. Intraoperative intravenous injection of indocyanine green (ICG) at 0.2 mg/kg guided the careful dissection of the splenic hilum and checked the spleen perfusion once the upper arterial branch was clamped. The subsequent wash-out of the ICG allowed inspection of the peripheral vascular return of the splenic remnant through polar veins. Surgery was uneventful with minimal blood loss. Follow-up ultrasound scan revealed a well-perfused small splenic remnant with no signs of recurrence. Laparoscopic partial splenectomy is feasible in benign splenic tumors, especially in those cases of peripheral location. Fluorescence facilitates the safe dissection of the splenic hilum, the visualization of the transection plane of the spleen and the perfusion of the remnant in cases of anatomically and technically complicated partial splenectomies.

First Experience with Fluorescence in Pediatric Laparoscopy.

Background The use of intraoperative fluorescence images with indocyanine green (ICG) has recently been described as an aid in decision-making during surgical procedures in adults. We present our first experiences with different laparoscopic procedures performed in children using ICG fluorescence images. Material and Method We have used ICG fluorescence imaging technique in varicocele ligation, two nephrectomies, cholecystectomy, and one case of aortocoronary fistula closure. All procedures were performed through a minimally invasive approach. A high definition camera equipped with a visible infrared light source and gray-scale vision technology was used. After injection of ICG before or during the laparoscopic procedure, precise identification of vascular anatomy and bile duct architecture were easily identified. Fluorescence helped to assess blood flow from the spermatic vessels, define the variability of renal vascularization, and determine the precise location of the aortocoronary fistula. Biliary excretion of the ICG allowed the definition of the biliary tract. Conclusion Fluorescein-assisted images allowed a clear definition of the anatomy and safe surgical maneuvers during surgical procedures. The ICG imaging system seems to be simple and safe. Larger and more specific studies are needed to confirm its applicability, expand its indications, and address its advantages and disadvantages.

Insights into the mechanism of paraoxonase-1: Comparing the reactivity of the six-bladed ß-propeller hydrolases.

The mammalian protein paraoxonase-1 (PON1) has been explored as a promising bioscavenger treatment for organophosphorus (OP) agent poisoning, but it is not active enough to protect against many agents. Engineering is limited because PON1's catalytic mechanism is poorly understood; moreover, its native activity and substrate are unknown. PON1 is a calcium-bound six-bladed ß-propeller hydrolase that shares high structural homology, a conserved metal-coordinating active site, and substrate specificity overlap with other members of a superfamily that includes squid diisopropylfluorophosphatase (DFPase), bacterial drug responsive protein 35 (Drp35), and mammalian senescence marker protein 30 (SMP30). We hypothesized that, by examining the reactivity of all four hydrolases using a common set of conservative mutations, we could gain further insight into the catalytic mechanism of PON1. We chose a set of mutations to examine conserved Asp and Glu residues in the hydrolase active sites, as well as the ligation sphere around the catalytic calcium and a His-His dyad seen in PON1. The wild-type (WT) and mutant hydrolases were assayed against a set of lactones, aryl esters, and OPs that PON1 is known to hydrolyze. Surprisingly, some mutations of Ca2+ coordinating residues, previously thought to be essential for turnover, resulted in significant activity toward all substrate classes examined. Additionally, merely maintaining WT-like charge in the active site of PON1 was insufficient for high activity. Finally, the H115-H134 dyad does not appear to be essential for catalysis against any substrate. Therefore, previously proposed mechanisms must be re-evaluated.

Assessing the stoichiometric efficacy of mammalian expressed paraoxonase-1 variant I-F11 to afford protection against G-type nerve agents.

We evaluated the ability of evolved paraoxonase-1 (PON1) to afford broad spectrum protection against G-type nerve agents when produced in mammalian cells via an adenovirus expression system. The PON1 variants G3C9, VII-D11, I-F11, VII-D2 and II-G1 were screened in vitro for their ability to hydrolyze G-agents, as well as for their preference towards hydrolysis of the more toxic P(-) isomer. I-F11, with catalytic efficiencies of (1.1 ± 0.1) × 106 M-1 min-1, (2.5 ± 0.1) × 106 M-1 min-1, (2.3 ± 0.5) × 107 M-1 min-1and (9.2 ± 0.1) × 106 M-1 min-1 against tabun (GA), sarin (GB), soman (GD) and cyclosarin (GF), respectively, was found to be a leading candidate for further evaluation. To demonstrate the broad spectrum efficacy of I-F11 against G-agents, a sequential 5 × LD50 dose of GD, GF, GB and GA was administered to ten mice expressing I-F11 on days 3, 4, 5 and 6 following virus injection, respectively. At the conclusion of the experiment, 80% of the animals survived exposure to all four G-agents. Using the concept of stoichiometric efficacy, we determined that I-F11 affords protection from lethality against an administered dose of 10, 15, 90 and 80 molar equivalents of GA, GB, GD and GF, respectively, relative to the molar equivalents of I-F11 in circulation. It also appears that I-F11 can associate with high density lipoprotein in circulation, suggesting that I-F11 retained this function of native PON1. This combination of attractive attributes demonstrates that I-F11 is an attractive candidate for development as a broad-therapeutic against G-type nerve agent exposure.

Retinal ganglion cell neuroprotection induced by activation of alpha7 nicotinic acetylcholine receptors.

The α7nAChR agonist, PNU-282987, has previously been shown to have a neuroprotective effect against loss of retinal ganglion cells (RGCs) in an in vivo glaucoma model when the agent was injected into the vitreous chamber of adult Long Evans rat eyes. Here, we characterized the neuroprotective effect of PNU-282987 at the nerve fiber and retinal ganglion cell layer, determined that neuroprotection occurred when the agonist was applied as eye drops and verified detection of the agonist in the retina, using LC/MS/MS. To induce glaucoma-like conditions in adult Long Evans rats, hypertonic saline was injected into the episcleral veins to induce scar tissue and increase intraocular pressure. Within one month, this procedure produced significant loss of RGCs compared to untreated conditions. RGCs were quantified after immunostaining with an antibody against Thy 1.1 and imaged using a confocal microscope. In dose-response studies, concentrations of PNU-282987 were applied to the animal's right eye two times each day, while the left eye acted as an internal control. Eye drops of PNU-282987 resulted in neuroprotection against RGC loss in a dose-dependent manner using concentrations between 100 µM and 2 mM PNU-282987. LC/MS/MS results demonstrated that PNU-282987 was detected in the retina when applied as eye drops, relatively small amounts of PNU-282987 were measured in blood plasma and no PNU-282987 was detected in cardiac tissue. These results support the hypothesis that eye drop application of PNU-282987 can prevent loss of RGCs associated with glaucoma, which can lead to neuroprotective treatments for diseases that involve α7nAChRs.

An in vitro and in vivo evaluation of the efficacy of recombinant human liver prolidase as a catalytic bioscavenger of chemical warfare nerve agents.

In this study, we determined the ability of recombinant human liver prolidase to hydrolyze nerve agents in vitro and its ability to afford protection in vivo in mice. Using adenovirus containing the human liver prolidase gene, the enzyme was over expressed by 200- to 300-fold in mouse liver and purified to homogeneity by affinity and gel filtration chromatography. The purified enzyme hydrolyzed sarin, cyclosarin and soman with varying rates of hydrolysis. The most efficient hydrolysis was with sarin, followed by soman and by cyclosarin {apparent kcat/Km [(1.9 ± 0.3), (1.7 ± 0.2), and (0.45 ± 0.04)] × 10(5 )M(-1 )min(-1), respectively}; VX and tabun were not hydrolyzed by the recombinant enzyme. The enzyme hydrolyzed P (+) isomers faster than the P (-) isomers. The ability of recombinant human liver prolidase to afford 24 hour survival against a cumulative dose of 2 × LD50 of each nerve agent was investigated in mice. Compared to mice injected with a control virus, mice injected with the prolidase expressing virus contained (29 ± 7)-fold higher levels of the enzyme in their blood on day 5. Challenging these mice with two consecutive 1 × LD50 doses of sarin, cyclosarin, and soman resulted in the death of all animals within 5 to 8 min from nerve agent toxicity. In contrast, mice injected with the adenovirus expressing mouse butyrylcholinesterase, an enzyme which is known to afford protection in vivo, survived multiple 1 × LD50 challenges of these nerve agents and displayed no signs of toxicity. These results suggest that, while prolidase can hydrolyze certain G-type nerve agents in vitro, the enzyme does not offer 24 hour protection against a cumulative dose of 2 × LD50 of G-agents in mice in vivo.

Investigation of evolved paraoxonase-1 variants for prevention of organophosphorous pesticide compound intoxication.

We investigated the ability of the engineered paraoxonase-1 variants G3C9, VII-D11, I-F11, and VII-D2 to afford protection against paraoxon intoxication. Paraoxon is the toxic metabolite of parathion, a common pesticide still in use in many developing countries. An in vitro investigation showed that VII-D11 is the most efficient variant at hydrolyzing paraoxon with a kcat/Km of 2.1 × 10(6) M(-1) min(-1) and 1.6 × 10(6) M(-1) min(-1) for the enzyme expressed via adenovirus infection of 293A cells and mice, respectively. Compared with the G3C9 parent scaffold, VII-D11 is 15- to 20-fold more efficacious at hydrolyzing paraoxon. Coinciding with these results, mice expressing VII-D11 in their blood survived and showed no symptoms against a cumulative 6.3 × LD50 dose of paraoxon, whereas mice expressing G3C9 experienced tremors and only 50% survival. We then determined whether VII-D11 can offer protection against paraoxon when present at substoichiometric concentrations. Mice containing varying concentrations of VII-D11 in their blood (0.2-4.1 mg/ml) were challenged with doses of paraoxon at fixed stoichiometric ratios that constitute up to a 10-fold molar excess of paraoxon to enzyme (1.4-27 × LD50 doses) and were assessed for tremors and mortality. Mice were afforded complete asymptomatic protection below a paraoxon-to-enzyme ratio of 8:1, whereas higher ratios produced tremors and/or mortality. VII-D11 in mouse blood coeluted with high-density lipoprotein, suggesting an association between the two entities. Collectively, these results demonstrate that VII-D11 is a promising candidate for development as a prophylactic catalytic bioscavenger against organophosphorous pesticide toxicity.

A nicotinic acetylcholine receptor agonist prevents loss of retinal ganglion cells in a glaucoma model.

PURPOSE: The purpose of this study was to analyze the neuroprotective effect of an α7 nAChR agonist, PNU-282987, using an in vivo model of glaucoma in Long Evans rats. METHODS: One eye in each animal was surgically manipulated to induce glaucoma in control untreated animals and in animals that were treated with intravitreal injections of PNU-282987. To induce glaucoma-like conditions, 0.05 mL of 2 M NaCl was injected into the episcleral veins of right eyes in each rat to create scar tissue and increase intraocular pressure. The left eye in each rat acted as an internal control. One month following NaCl injection, rats were euthanized, retinas were removed, flatmounted, fixed, and nuclei were stained with cresyl violet or RGCs were immunostained with an antibody against Thy 1.1 or against Brn3a. Stained nuclei in the RGC layer and labeled RGCs in NaCl-injected retinas were counted and compared with cell counts from untreated retinas in the same animal. RESULTS: NaCl injections into the episcleral veins caused a significant loss of cells by an average of 27.35% (± 2.12 SEM) in the RGC layer within 1 month after NaCl injection, which corresponded to a significant loss of RGCs. This loss of RGCs was eliminated if 5 µL of 100 µM PNU-282987 was injected into the right eye an hour before NaCl injection. CONCLUSIONS: The results from this study support the hypothesis that the α7 agonist, PNU-282987, has a neuroprotective effect in the rat retina. PNU-282987 may be a viable candidate for future therapeutic treatments of glaucoma.

Meconium aspiration syndrome, parental atopy and asthma symptoms in children under two years old.

OBJECTIVE: To determine the association among meconium aspiration syndrome, parental atopy and asthma symptoms in children younger than two years. MATERIAL AND METHODS: One hundred thirty six children who had suffered meconium aspiration syndrome were followed from birth to they were two years old. CONTROL GROUP included 136 healthy children without meconium aspiration syndrome. RESULTS: Group of children with meconium aspiration syndrome: The average hospital stay in neonatal intensive care unit was of 5.91 +/- 4.44 days and were given 65.5 +/- 16.6 hours of oxygen. Prevalence of asthma symptoms during the last year was of 41.2%; 35.7% had family history of allergies (p = NS). The average age at the beginning of asthma symptoms was of 12.64 +/- 6.96 months; 78.6% of children were male. CONTROL GROUP: 30.9% had wheezing episodes during the last 12 months; 28.6% were male; 38.1% had family history of allergies (p = NS). The average age at the beginning of asthma symptoms was of 15.57 +/- 6.05 months. CONCLUSION: Aspiration of meconium seems to be an important risk factor of the early beginning of asthma symptoms in children younger than two years. Data shows that the prevalence of asthma symptoms was higher in case group than in control group. Asthma symptoms risk increases in children with aspiration meconium syndrome and family background of allergies.