PAM-free diagnostics with diverse type V CRISPR-Cas systems.

Type V CRISPR-Cas effectors have revolutionized molecular diagnostics by facilitating the detection of nucleic acid biomarkers. However, their dependence on the presence of protospacer adjacent motif (PAM) sites on the target double-stranded DNA (dsDNA) greatly limits their flexibility as diagnostic tools. Here we present a novel method named PICNIC that solves the PAM problem for CRISPR-based diagnostics with just a simple ∼10-min modification to contemporary CRISPR-detection protocols. Our method involves the separation of dsDNA into individual single-stranded DNA (ssDNA) strands through a high- temperature and high-pH treatment. We then detect the released ssDNA strands with diverse Cas12 enzymes in a PAM-free manner. We show the utility of PICNIC by successfully applying it for PAM-free detection with three different subtypes of the Cas12 family- Cas12a, Cas12b, and Cas12i. Notably, by combining PICNIC with a truncated 15-nucleotide spacer containing crRNA, we demonstrate PAM-independent detection of clinically important single- nucleotide polymorphisms with CRISPR. We apply this approach to detect the presence of a drug-resistant variant of HIV-1, specifically the K103N mutant, that lacks a PAM site in the vicinity of the mutation. Additionally, we successfully translate our approach to clinical samples by detecting and genotyping HCV-1a and HCV-1b variants with 100% specificity at a PAM-less site within the HCV genome. In summary, PICNIC is a simple yet groundbreaking method that enhances the flexibility and precision of CRISPR-Cas12-based diagnostics by eliminating the restriction of the PAM sequence.

Antidepressant mechanisms of ketamine: a review of actions with relevance to treatment-resistance and neuroprogression.

Concurrent with recent insights into the neuroprogressive nature of depression, ketamine shows promise in interfering with several neuroprogressive factors, and has been suggested to reverse neuropathological patterns seen in depression. These insights come at a time of great need for novel approaches, as prevalence is rising and current treatment options remain inadequate for a large number of people. The rapidly growing literature on ketamine's antidepressant potential has yielded multiple proposed mechanisms of action, many of which have implications for recently elucidated aspects of depressive pathology. This review aims to provide the reader with an understanding of neuroprogressive aspects of depressive pathology and how ketamine is suggested to act on it. Literature was identified through PubMed and Google Scholar, and the reference lists of retrieved articles. When reviewing the evidence of depressive pathology, a picture emerges of four elements interacting with each other to facilitate progressive worsening, namely stress, inflammation, neurotoxicity and neurodegeneration. Ketamine acts on all of these levels of pathology, with rapid and potent reductions of depressive symptoms. Converging evidence suggests that ketamine works to increase stress resilience and reverse stress-induced dysfunction, modulate systemic inflammation and neuroinflammation, attenuate neurotoxic processes and glial dysfunction, and facilitate synaptogenesis rather than neurodegeneration. Still, much remains to be revealed about ketamine's antidepressant mechanisms of action, and research is lacking on the durability of effect. The findings discussed herein calls for more longitudinal approaches when determining efficacy and its relation to neuroprogressive factors, and could provide relevant considerations for clinical implementation.

Maternal exercise during pregnancy modulates mitochondrial function and redox status in a sex-dependent way in adult offspring's skeletal muscle.

Maternal exercise has shown beneficial effects on mother and child. Literature confirm progeny's cognition improvement, and upregulation in neurotrophins, antioxidant network, and DNA repair system. Considering that there is a lack of information demonstrating the impact of maternal exercise on offspring's skeletal muscle, we aimed to investigate the mitochondrial and redox effects elicited by maternal swimming. Adult female Wistar rats were divided into three groups: control sedentary, free swimming, and swimming with overload (2% of the body weight). Exercised groups were submitted weekly to five swimming sessions (30 min/day), starting 1 week prior to the mating and lasting to the delivery. Gastrocnemius and soleus muscle from 60-day-old offspring were analyzed. Our results clearly showed a sex-dependent effect. Male soleus showed increased mitochondrial functionality in the overload group. Female muscle from the overload group adapted deeply. Considering the redox status, the female offspring delivered to overload exercised dams presented reduced oxidants levels and protein damage, allied to downregulated antioxidant defenses. We also observed an increase in the mitochondrial function in the gastrocnemius muscle of the female offspring born from overload exercised dams. Soleus from female delivered to the overload exercise group presented reduced mitochondrial activity, as well as reduced reactive species, protein carbonyls, and antioxidant network, when compared to the male. In conclusion, maternal exercise altered the redox status and mitochondrial function in the offspring's skeletal muscle in a sex-dependent way. The clinical implication was not investigated; however, the sexual dimorphism in response to maternal exercise might impact exercise resilience in adulthood.

Developmental programming: intrauterine caloric restriction promotes upregulation of mitochondrial sirtuin with mild effects on oxidative parameters in the ovaries and testes of offspring.

According to the developmental origins of health and disease (DOHaD) hypothesis, changes in the maternal environment are known to reprogram the metabolic response of offspring. Known for its redox modulation, caloric restriction extends the lifespan of some species, which contributes to diminished cellular damage. Little is known about the effects of gestational caloric restriction, in terms of antioxidant parameters and molecular mechanisms of action, on the reproductive organs of offspring. This study assessed the effects of moderate (20%) caloric restriction on redox status parameters, molecular expression of sirtuin (SIRT) 1 and SIRT3 and histopathological markers in the ovaries and testes of adult rats that were subjected to gestational caloric restriction. Although enzyme activity was increased, ovaries from female pups contained high levels of oxidants, whereas testes from male pups had decreased antioxidant enzyme defences, as evidenced by diminished glyoxalase I activity and reduced glutathione content. Expression of SIRT3, a deacetylase enzyme related to cellular bioenergetics, was increased in both ovaries and testes. Previous studies have suggested that, in ovaries, diminished antioxidant metabolism can lead to premature ovarian failure. Unfortunately, there is little information regarding the redox profile in the testis. This study is the first to assess the redox network in both ovaries and testes, suggesting that, although intrauterine caloric restriction improves molecular mechanisms, it has a negative effect on the antioxidant network and redox status of reproductive organs of young adult rats.

Effect of Maternal Exercise on Diet-induced Redox Imbalance in Hippocampus of Adult Offspring.

Physical exercise practice has been increasingly recommended in the prevention and treatment of chronic diseases, causing a positive effect from body weight/fat loss to improved cognitive function. Maternal exercise seems to induce the same positive lifelong adaptations to the offspring. We hypothesized that maternal exercise can prevent redox imbalance in adult offspring's hippocampus exposed to a high-fat diet (HFD). Female Wistar rats were divided into three groups before and during pregnancy: (1) sedentary, (2) swimming exercise, and (3) swimming exercise with overload. On 60 days of age, the male pups were divided into standard diet or HFD for one month, yielding normal and HFD subgroups for each maternal condition. Maternal interventions did not alter gestational parameters, birth outcomes, and offspring weight gain from weaning to 90 days of age. The HFD consumption increased body fat, which was not prevented by maternal exercise. Serum glucose levels were increased by HFD, an effect that was prevented by unload maternal exercise. In the hippocampus, both maternal exercise intensities could increase antioxidant defense. Hippocampal redox homeostasis was impaired by HFD, causing increased superoxide levels, which was prevented by exercise without load, while overload caused only a reduction of the effect. In summary, the practice of swimming exercise without overload during pregnancy seems to be more beneficial when evaluated in animal model, preventing HFD induced redox imbalance and increasing antioxidant defense while overload swimming exercise during pregnancy demonstrated a negative effect on offspring submitted to HFD consumption.

Serum carotenoids and macular pigment optical density in patients with intestinal resections and healthy subjects: an exploratory study.

Reduced absorption capacity in patients with intestinal resections (IR) could result in malabsorption of fat-soluble components like carotenoids, which are of clinical interest in relation to visual health. In this case cohort, we investigated the association between IR and serum lutein, zeaxanthin, ß-carotene and macular pigment optical density, when compared with healthy controls. Ten patients with IR and twelve healthy controls were included in the study. Baseline characteristics were comparable between groups, except for higher serum TAG (P < 0·05) and shorter bowel length (P < 0·0001) in the group with IR. Serum lutein, zeaxanthin, ß-carotene and macular pigment optical density were >15 % lower in the patient group compared with healthy controls (P < 0·05, adjusted for age) and, in the case of serum lutein and zeaxanthin, also for dietary intake of carotenoids. Results suggest that for a test of macular carotenoid supplementation, subjects with a potentially clinically significant carotenoid deficit could be recruited among patients with IR.

The use of metabolic balance studies in the objective discrimination between intestinal insufficiency and intestinal failure.

Background: In research settings that use metabolic balance studies (MBSs) of stable adult patients with short bowel syndrome, intestinal failure (IF) and dependence on parenteral support (PS) have been defined objectively as energy absorption <84% of calculated basal metabolic rate (BMR), wet weight (WW) absorption <23 g · kg body weight-1 · d-1, or both. Objective: This study aimed to explore and validate these borderlines in the clinical setting.Design: Intestinal absorption was measured from April 2003 to March 2015 in 175 consecutive patients with intestinal insufficiency (INS) in 96-h MBSs. They had not received PS 3 mo before referral.Results: To avoid the need for PS, the minimum absorptive requirements were energy absorption of ≥81% of BMR and WW absorption of ≥21 g · kg body weight-1 · d-1, which were equivalent to findings in research settings (differences of 3.6% and 8.7%; P = 0.65 and 0.60, respectively). Oral failure defined as energy intake <130% of calculated BMR or WW intake <40 g · kg body weight-1 · d-1 was seen in 71% and 82% of the 10% of patients with the lowest energy absorption and WW absorption, respectively.Conclusions: In clinical settings, the borderlines between INS and IF were not significantly different from those in research settings, even in an unselected patient population in which oral failure was also a predominant cause of nutritional dyshomeostasis. MBSs may be recommended to identify the individual patient in the spectrum from INS to IF, to objectivize the cause of nutritional dyshomeostasis (oral failure, malabsorption, or both), and to quantify the effects of treatment.

Polyols accumulated in ribose-5-phosphate isomerase deficiency increase mitochondrial superoxide production and improve antioxidant defenses in rats' prefrontal cortex.

The ribose-5-phosphate isomerase deficiency is an inherited condition, which results in cerebral d-arabitol and ribitol accumulation. Patients present leukoencephalopathy, mental retardation, and psychomotor impairment. Considering that the pathophysiology of this disorder is still unclear, and literature are sparse and contradictory, reporting pro and antioxidant activities of polyols, the main objective of this study was to investigate some parameters of oxidative homeostasis of prefrontal cortex of rats incubated with d-arabitol and ribitol. We found evidences that ribitol promoted an increase in antioxidant enzymes activity (superoxide dismutase, catalase, and glutathione peroxidase), probably secondary to enhanced production of superoxide radical, measured by flow cytometry. Oxidation of proteins and lipids was not induced by polyols. Our data allow us to conclude that, at least in our methodological conditions, arabitol and ribitol probably have a secondary effect on the pathophysiology of ribose-5-phosphate isomerase deficiency.

Maintaining a way of life for São Miguel Island (the Azores archipelago, Portugal): an assessment of coastal processes and protection.

Traditional hard engineering structures and recently emerging soft engineering alternatives have been employed to protect vulnerable coastlines. Despite negative publicity, they have ensured community survival where socio-economic benefits outweigh adverse impacts. This is especially true for Small Islands (SI) where increasing sea levels and storm intensities threaten already limited land availability. This paper presents coastal vulnerability in São Miguel Island (the Azores SI archipelago) and considers SI issues with regard to coastal land loss. Regional wave statistics using 1998 to 2011 wind record showed: periods ranging from 7 to 13s (circa 83%); wave heights between 1 and 3m (circa 60%); and increasing trends in westerly (p=0.473), easterly (p=0.632) and southeasterly (p=0.932) waves. Sea level analyses between 1978 and 2007 indicated a statistically significant rising trend (2.5 ± 0.4 mm yr(-1); p=0.000), while between 1996 and 2007 it was 3.3 ± 1.5 mm yr(-1) (p=0.025), agreeing with other global sea level studies. Based on 2001 and 2008 population data and using zonal statistics, circa 60% of the Island's population was found to reside within 1 km of the sea and the percentage of total population was linearly correlated with distance from the shoreline (r(2)=99%). Three case studies show hard coastal engineering solutions preserved Azorean coastal lifestyle and had little or no observed negative impacts on their environs. Although hard engineering is likely to remain a valuable and feasible coastal protection option, an inventory of São Miguel's population distribution, surf breaks, bathymetry and coastal erosion rates showed the potential of using multifunctional artificial reefs as a soft engineering solution. These offshore submerged breakwaters offer coastal protection while providing additional benefits such as surfing amenity and beach widening. Consequently, findings of this work can inform other SI communities.

Favre-Racouchot syndrome: a novel two-step treatment approach using the carbon dioxide laser.

BACKGROUND: Favre-Racouchot syndrome (FRS) is both disfiguring and difficult to treat. Available medical and surgical therapies are of variable efficacy. Most treatments do not achieve complete resolution and do not show maintenance of therapeutic response. OBJECTIVES: To assess the response to a novel two-step treatment using the CO2 laser in patients with FRS. METHODS: Seven patients with FRS were treated with the CO2 laser in resurfacing mode with manual expression of comedones under infiltrative local anaesthesia. The procedure was completed in one treatment session lasting 30 min and the wound was left to heal by secondary intention. A topical antibiotic was applied to treated areas, which were covered with a nonadherent dressing. All patients were assessed 3 months postoperatively by the operating laser surgeon and a visual assessment of clinical response to treatment in comparison with pretreatment photographs was made. Patient satisfaction was also recorded. RESULTS: All patients achieved complete resolution of FRS. The follow-up duration for our cohort ranged from 8 months to 3 years. Two patients required further treatment within a 2-3-year period from initial treatment. Disease relapse was noted over 1 year after the primary treatment; both these cases were smokers and repeat treatment with similar laser parameters maintained reproducible results. Our longest disease-free follow-up duration was 3 years postprimary treatment. The laser surgeons and patients reported high levels of therapeutic benefit and satisfaction with the results. CONCLUSIONS: This two-step treatment of FRS (CO2 laser resurfacing and manual pressure-induced expression of comedones) is an effective and durable treatment for FRS with an excellent cosmetic outcome. Long-term follow-up beyond 3 years is planned to determine whether later recurrence occurs with this technique.

A study of the efficacy of carbon dioxide and pigment-specific lasers in the treatment of medium-sized congenital melanocytic naevi.

BACKGROUND: Treatment of medium-sized congenital melanocytic naevi (CMN) can be challenging. OBJECTIVES: To present the results of treatment of 55 CMN with the carbon dioxide (CO(2) ) and pigment-specific lasers. METHOD: CO(2) and Q-switched lasers (frequency-doubled Nd:YAG, Nd:YAG and alexandrite) were used to treat 55 CMN. Patients were treated at 3-month intervals until maximum clearance. Clinical response at 3-6 months after final treatment was graded as poor (< 50%), good (50-75%) or excellent (> 75%). Outcomes were evaluated on case note review and questionnaire. RESULTS: Thirty-six of the 55 CMN were macular and 19 were mammillated. Twenty-seven CMN were present on the head and neck. For macular CMN, outcomes were better for truncal CMN. Scarring and pallor were seen in three lower limb macular CMN treated with a CO(2) laser. Mammillated CMN on the head and neck showed most improvement. Pigment-specific lasers were of no additional benefit. Repigmentation occurred in 6% of macular and 21% of mammillated CMN. Partial or complete regimentation of CMN was reported by 46% of patients. CONCLUSIONS: Compared with macular CMN, mammillated CMN show a marginally better response to laser treatment. CMN on the limbs respond poorly. Pigment-specific lasers do not lighten mammillated CMN. Adverse effects can occur with CO(2) laser treatment of macular CMN on lower limbs.

Efficacy of topical tacrolimus 0.3% in clobetasol propionate 0.05% ointment in therapy-resistant cutaneous lupus erythematosus: a cohort study.

BACKGROUND: Despite a range of available topical and systemic therapies, treatment of cutaneous lupus erythematosus (CLE) can be challenging. Objectives. To evaluate the efficacy of a specially formulated preparation of tacrolimus 0.3% in clobetasol propionate 0.05% ointment (TCPO) in the treatment of CLE. METHODS: Case notes of 13 patients with treatment-resistant CLE (11 discoid LE, 1 systemic LE and 1 subacute cutaneous LE) who had used twice-daily TCPO (TCPO group) were reviewed. These were compared with five similar patients with resistant CLE who had been given 0.1% tacrolimus ointment alone (TO group). RESULTS: In the TCPO group (mean treatment duration 20 months, range 1-72), a good or excellent response was seen in five and six patients, respectively; one patient showed slight improvement. Telangiectasia and acne were observed in two patients. No systemic side-effects were noted. In the TO group (mean treatment duration 6 months, range 1-24), one patient showed good improvement and two showed slight improvement. CONCLUSION: The results of our small retrospective study suggest that TCPO may be more effective than either 0.1% tacrolimus or clobetasol propionate 0.05% ointment monotherapy in the treatment of recalcitrant CLE. Randomized controlled trials are needed to confirm these preliminary findings.

Carbon dioxide laser treatment of rhinophyma: a review of 124 patients.

BACKGROUND: Rhinophyma is a progressive, localized or generalized nasal deformity resulting from hypertrophy of sebaceous and connective tissue. The CO(2) laser has been used for treatment of rhinophyma, but the long-term efficacy of the treatment is unknown. OBJECTIVES: To review the outcome of 124 patients with rhinophyma treated with the CO(2) laser between 1996 and 2008 in our centre. PATIENTS AND METHODS: Exuberant sebaceous tissue was ablated using the Sharplan 40C CO(2) laser (Sharplan Lasers UK Ltd, London, U.K.) under local anaesthesia. The technique varied with the severity of rhinophyma; the laser was used in a continuous mode to debulk the larger rhinophymas, and in a resurfacing mode (Silk Touch scanner; Sharplan, 4-7-mm spot at 20-40 W) or continuous mode (10-20 W using a defocused 2-3-mm beam) to reshape the nasal contours. Outcomes were determined by case notes, clinical review and questionnaire. RESULTS: Laser treatment was completed in a single session in 115 of 124 patients. All patients were reviewed 3 months post-treatment. Results were classified as good to excellent in 118 and poor in six patients. All patients were sent a satisfaction questionnaire in 2008 and 52 patients replied. Patients reported high levels of satisfaction following treatment. The post-treatment response at 3-month review was maintained long term. The main complications were pain associated with injection of local anaesthetic, scarring and hypopigmentation (four patients) and open pores (two patients). CONCLUSIONS: The CO(2) laser is an effective and durable treatment for rhinophyma. Treatment carries a low risk of side-effects and is associated with high patient acceptability and satisfaction.

Pulsed-dye laser treatment of angioma serpiginosum.

Angioma serpiginosum (AS) is a rare, benign, vascular disorder comprising nonblanchable punctate red to purple lesions in a gyrate or serpiginous configuration. Tunable pulsed-dye laser (PDL) therapy has been successfully used in the treatment of AS. We report our results of treatment of 12 AS sites in eight patients who were treated with PDL. After an average of 3.75 treatments, complete resolution of AS was seen in five lesions, an excellent response in four and a good response in three. Self-limiting hyperpigmentation at the test sites was noted in two patients. This study provides further evidence of the efficacy of PDL for treatment of AS.

Outcomes of treatment of nine cases of recalcitrant severe hidradenitis suppurativa with carbon dioxide laser.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic and often a recalcitrant inflammatory skin condition. OBJECTIVES: To present the results of carbon dioxide (CO2) laser treatment of recalcitrant HS in nine patients who had failed to improve on medical and other surgical treatments. METHODS: HS lesions consisting of abscesses, sinuses and granulation tissue were completely excised using the cutting mode of a CO2 laser, leaving only healthy residual subcutaneous fat. The wounds were closed by primary intention where possible and left to granulate otherwise. Outcomes were determined by clinical review and questionnaire. RESULTS: Twenty-seven sites were treated in 19 sessions on nine patients. Seven procedures were performed under general anaesthesia and 12 under local. All patients rated their postoperative discomfort as less or equal to their preoperative state. Seven of the nine patients had complete remission for 12 months or longer after their last laser treatment and ceased all medications. High levels of patient satisfaction were reported with CO2 laser treatment. The main complication was axillary scar contracture in two patients but this was insufficient to limit limb movement. CONCLUSIONS: CO2 laser treatment should be considered as a treatment option in recalcitrant HS, where multiple medical treatments have been ineffective.

Synthesis and preclinical evaluation of a folic acid derivative labeled with 18F for PET imaging of folate receptor-positive tumors.

UNLABELLED: Folic acid was linked regioselectively through its alpha- and gamma-carboxyl groups to 4-fluorobenzylamine (FBA), and the alpha- and gamma-FBA-folate regioisomers were evaluated for their ability to bind to folate receptor-positive cells. The 18F-labeled alpha/gamma-FBA-folate counterpart was examined for in vivo tumor targeting efficiency in nude mice bearing folate receptor-positive tumor cells. METHODS: 18F-alpha/gamma-FBA-folate was prepared in a 4-step reaction sequence starting from folic acid. The relative binding affinities of the alpha- and gamma-FBA-folates to the folate receptor with respect to parent folic acid were determined in cultured KB-31 cells (nasopharyngeal epidermal carcinoma cell line) overexpressing the folate receptor using 3H-folic acid. Tumor accumulation of the 18F-labeled alpha/gamma-FBA-folate and 18F-FDG was analyzed in vivo by high-resolution PET. Biodistribution and PET studies were performed under baseline and blockage conditions. RESULTS: The radiochemical yield of the coupling step ranged from 15% to 44%, and the maximum specific radioactivity was 24 GBq/micromol. The in vitro binding affinities of the alpha- and gamma-isomers and folic acid were 71, 62, and 41 nmol/L, respectively. PET revealed heterogeneous uptake of the radioligand, with the highest activity concentrations found in the tumor rim. In contrast, 18F-FDG uptake in a nude mouse bearing KB-31 folate receptor-positive tumors was negligible. Radioligand uptake in tumors at 125 min after injection amounted to 6.56% of the injected dose per gram of tissue (%ID/g) in control animals, whereas radioactivity accumulation in the tumors of folic acid-treated animals was significantly reduced by more than 80%-to 1.07 %ID/g (P = 0.001). CONCLUSION: This new 18F-labeled folic acid derivative is a promising tool for PET imaging of folate receptor-positive tumors.