Full-aperture extended-depth oblique plane microscopy through dynamic remote focusing.

Significance: The reprojection setup typical of oblique plane microscopy (OPM) limits the effective aperture of the imaging system, and therefore its efficiency and resolution. Large aperture system is only possible through the use of custom specialized optics. A full-aperture OPM made with off the shelf components would both improve the performance of the method and encourage its widespread adoption. Aim: To prove the feasibility of an OPM without a conventional reprojection setup, retaining the full aperture of the primary objective employed. Approach: A deformable lens based remote focusing setup synchronized with the rolling shutter of a complementary metal-oxide semiconductor detector is used instead of a traditional reprojection system. Results: The system was tested on microbeads, prepared slides, and zebrafish embryos. Resolution and pixel throughput were superior to conventional OPM with cropped apertures, and comparable with OPM implementations with custom made optical components. Conclusions: An easily reproducible approach to OPM imaging is presented, eliminating the conventional reprojection setup and exploiting the full aperture of the employed objective.

Cytisine as a smoking cessation aid: Preliminary observations with a modified therapeutic scheme in real life.

INTRODUCTION: Cigarette smoke accounts for over 90,000 deaths each year in Italy. Tobacco dependence treatment guidelines suggest adopting an integrated pharmacological-behavioral model of intervention. Cytisine is a partial agonist of nicotinic receptors. Trials conducted to date have demonstrated its good efficacy in promoting smoking cessation. The cytisine scheme of treatment consists of 25 days of treatment. A 40-day regimen, with an escalating dose and an extended duration of the treatment, has been in use in many anti-smoking centers in Italy for several years, but to date there are no reports on the use of cytisine with this scheme. METHODS: A retrospective, real-life, observational study was conducted between January 2016 and September 2022. The 300 patients who had received at least one dose of study medication were selected. Continuous variables were compared by the Wilcoxon-Mann-Whitney test. Univariate and multivariate logistic regression models were implemented for self-reported seven-day point prevalence for abstinence at three, six and 12 months. RESULTS: The median age of the patients was 59 years, 57% were women. The median smoking exposure was 33.8 pack-years. Self-reported smoking abstinence at three, six and 12 months was 68.7%, 56.3% and 47.3% respectively. 84% completed the cytisine treatment, 31.3% reported adverse events and in 8.3% these led to dropping out of the treatment. CONCLUSION: Cytisine, administered with a novel therapeutic scheme in the real-life setting of a specialized anti-smoking center, significantly promotes smoking abstinence. However, more studies are needed to assess the tolerability and efficacy of this new regimen.

Low environmental impact remediation of microplastics: Visible-light photocatalytic degradation of PET microplastics using bio-inspired C,N-TiO2/SiO2 photocatalysts.

Microplastics (MPs) are plastic particles with sizes between 1 µm and 5 mm with a ubiquitous presence in aquatic ecosystems. MPs harm marine life and can cause severe health problems for humans. Advanced oxidation processes (AOPs) that involve the in-situ generation of highly oxidant hydroxyl radicals can be an alternative to fight MPs pollution. Of all the AOPs, photocatalysis has been proven a clean technology to overcome microplastic pollution. This work proposes novel C,N-TiO2/SiO2 photocatalysts with proper visible-active properties to degrade polyethylene terephthalate (PET) MPs. Photocatalysis was performed in an aqueous medium and at room temperature, evaluating the influence of two pH values (pH 6 and 8). The results demonstrated that the degradation of the PET MPs by C,N-TiO2/SiO2 semiconductors is possible, achieving mass losses between 9.35 and 16.22 %.

Local aberration control to improve efficiency in multiphoton holographic projections.

Optical aberrations affect the quality of light propagating through a turbid medium, where refractive index is spatially inhomogeneous. In multiphoton optical applications, such as two-photon excitation fluorescence imaging and optogenetics, aberrations non-linearly impair the efficiency of excitation. We demonstrate a sensorless adaptive optics technique to compensate aberrations in holograms projected into turbid media. We use a spatial light modulator to project custom three dimensional holographic patterns and to correct for local (anisoplanatic) distortions. The method is tested on both synthetic and biological samples to counteract aberrations arising respectively from misalignment of the optical system and from samples inhomogeneities. In both cases the anisoplanatic correction improves the intensity of the stimulation pattern at least two-fold.

Bacterial polyarthritis in post-weaning pigs in a high-density swine breeding area in Italy.

We assessed the bacterial agents found in 8-12-wk-old post-weaning pigs with arthritis. The bodies of 178 post-weaning pigs from 90 farms (average of 2 pigs/farm) with recurrent problems of lameness and swollen joints in a high-density breeding area were submitted for autopsy and sampled for further bacterial investigation. The most common articular gross lesions and histopathologic findings were serofibrinous (95 of 178; 53%) or serous (65 of 178; 37%) arthritis; suppurative lesions were less frequent (18 of 178; 10%). In 133 of 178 (74.7%) cases, a bacterial agent was detected in joints. Mycoplasma hyorhinis was the most common bacterium detected (82 of 133; 61.6%). Haemophilus parasuis and Streptococcus spp. were observed in 27 of 133 (20.3%) and 24 of 133 (18.0%) cases, respectively. Other bacteria in the 113 cases, considered less important, in order of their low frequency, were Mycoplasma spp. (13; 9.8%), Trueperella pyogenes (11; 8.2%), Mycoplasma hyosynoviae (4; 3.0%), Staphylococcus spp. (3; 2.2%), Escherichia coli (2; 1.5%), and Actinobacillus spp. (2; 1.5%). Our results highlight the primary role of M. hyorhinis compared to other microorganisms involved in young pigs with arthritis.

Experimental determination of shift-less aberration bases for sensorless adaptive optics in nonlinear microscopy.

Adaptive optics can improve the performance of optical systems and devices by correcting phase aberrations. While in most applications wavefront sensing is employed to drive the adaptive optics correction, some microscopy methods may require sensorless optimization of the wavefront. In these cases, the correction is performed by describing the aberration as a linear combination of a base of influence functions, optimizing an image quality metric as a function of the coefficients. The influence functions base is generally chosen to either efficiently represent the adaptive device used or to describe generic wavefronts in an orthogonal fashion. A rarely discussed problem is that most correction bases have elements which introduce, together with a correction of the aberration, a shift of the imaging field of view in three dimensions. While simple methods to solve the problem are available for linear microscopy methods, nonlinear microscopy techniques such as multiphoton or second harmonic generation microscopy require non-trivial base determination. In this paper, we discuss the problem, and we present a method for calibrating a shift-less base on a spatial light modulator for two-photon microscopy.

Identification of Hepatitis E Virus Genotypes 3 and 7 in Israel: A Public Health Concern?

BACKGROUND: Hepatitis E (HEV) is an emerging cause of viral hepatitis worldwide. Swine carrying hepatitis E genotype 3 (HEV-3) are responsible for the majority of chronic viral hepatitis cases in developed countries. Recently, genotype 7 (HEV-7), isolated from a dromedary camel in the United Arab Emirates, was also associated with chronic viral hepatitis in a transplant recipient. In Israel, chronic HEV infection has not yet been reported, although HEV seroprevalence in humans is ~10%. Camels and swine are >65% seropositive. Here we report on the isolation and characterization of HEV from local camels and swine. METHODS: Sera from camels (n = 142), feces from swine (n = 18) and blood from patients suspected of hepatitis E (n = 101) were collected during 2017-2020 and used to detect and characterize HEV sequences. RESULTS: HEV-3 isolated from local swine and the camel-derived HEV-7 sequence were highly similar to HEV-3f and HEV-7 sequences (88.2% and 86.4%, respectively) related to viral hepatitis. The deduced amino acid sequences of both isolates were also highly conserved (>98%). Two patients were HEV-RNA positive; acute HEV-1 infection could be confirmed in one of them. DISCUSSION: The absence of any reported HEV-3 and HEV-7 infection in humans remains puzzling, especially considering the reported seroprevalence rates, the similarity between HEV sequences related to chronic hepatitis and the HEV genotypes identified in swine and camels in Israel.

Airborne Coronaviruses: Observations from Veterinary Experience.

The virus responsible for the pandemic that has affected 152 countries worldwide is a new strain of coronavirus (CoV), which belongs to a family of viruses widespread in many animal species, including birds, and mammals including humans. Indeed, CoVs are known in veterinary medicine affecting several species, and causing respiratory and/or enteric, systemic diseases and reproductive disease in poultry. Animal diseases caused by CoV may be considered from the following different perspectives: livestock and poultry CoVs cause mainly "population disease"; while in companion animals they are a source of mainly "individual/single subject disease". Therefore, respiratory CoV diseases in high-density, large populations of livestock or poultry may be a suitable example for the current SARS-CoV-2/COVID-19 pandemic. In this review we describe some strategies applied in veterinary medicine to control CoV and discuss if they may help to develop practical and useful strategies to control the SARS-CoV-2/COVID-19 pandemic.

Real Time Generation of Three Dimensional Patterns for Multiphoton Stimulation.

The advent of optogenetics has revolutionized experimental research in the field of Neuroscience and the possibility to selectively stimulate neurons in 3D volumes has opened new routes in the understanding of brain dynamics and functions. The combination of multiphoton excitation and optogenetic methods allows to identify and excite specific neuronal targets by means of the generation of cloud of excitation points. The most widely employed approach to produce the points cloud is through a spatial light modulation (SLM) which works with a refresh rate of tens of Hz. However, the computational time requested to calculate 3D patterns ranges between a few seconds and a few minutes, strongly limiting the overall performance of the system. The maximum speed of SLM can in fact be employed either with high quality patterns embedded into pre-calculated sequences or with low quality patterns for real time update. Here, we propose the implementation of a recently developed compressed sensing Gerchberg-Saxton algorithm on a consumer graphical processor unit allowing the generation of high quality patterns at video rate. This, would in turn dramatically reduce dead times in the experimental sessions, and could enable applications previously impossible, such as the control of neuronal network activity driven by the feedback from single neurons functional signals detected through calcium or voltage imaging or the real time compensation of motion artifacts.

Association Between Infectious Agents and Lesions in Post-Weaned Piglets and Fattening Heavy Pigs With Porcine Respiratory Disease Complex (PRDC).

Porcine Respiratory Disease Complex (PRDC) is a multifactorial syndrome that causes health problems in growing pigs and economic losses to farmers. The etiological factors involved can be bacteria, viruses, or mycoplasmas. However, environmental stressors associated with farm management can influence the status of the animal's health. The role and impact of different microorganisms in the development of the disease can be complex, and these are not fully understood. The severity of lesions are a consequence of synergism and combination of different factors. The aim of this study was to systematically analyse samples, conferred to the Veterinary Diagnostic Laboratory (IZSLER, Brescia), with a standardized diagnostic protocol in case of suspected PRDC. During necropsy, the lungs and carcasses were analyzed to determine the severity and extension of lesions. Gross lung lesions were classified according to a pre-established scheme adapted from literature. Furthermore, pulmonary, pleural, and nasal lesions were scored to determine their severity and extension. Finally, the presence of infectious agents was investigated to identify the microorganisms involved in the cases studied. During the years 2014-2016, 1,658 samples of lungs and carcasses with PRDC from 863 farms were analyzed; among them 931 and 727 samples were from weaned piglets and fattening pigs, respectively. The most frequently observed lesions were characteristic of catarrhal bronchopneumonia, broncho-interstitial pneumonia, pleuropneumonia, and pleuritis. Some pathogens identified were correlated to specific lesions, whereas other pathogens to various lesions. These underline the need for the establishment of control and treatment programmes for individual farms.

Causes of swine polyserositis in a high-density breeding area in Italy.

We assessed the causes of polyserositis in pigs, categorized by causative agents and ages of animals affected. In a 3-y study, 246 pigs from 80 different farms with recurrent problems of polyserositis, in a high-density breeding area, were submitted for autopsy; 154 pigs with typical fibrinous serosal lesions were sampled for further bacterial and viral investigation. The most common gross lesions were pleuritis and pericarditis (141 of 154; 92%). The animals most affected were weaned pigs (139 of 154; 90%). Haemophilus parasuis and Mycoplasma hyorhinis were the most common bacteria detected and were present at the same rate (85 of 154; 55%). Other bacteria isolated were Streptococcus sp. (44 of 154; 29%), Pasteurella multocida (21 of 154; 14%), Escherichia coli (19 of 154; 12%), Actinobacillus pleuropneumoniae (7 of 154; 5%), and Trueperella pyogenes (4 of 154; 3%). Porcine reproductive and respiratory syndrome virus (PRRSV; 119 of 154; 77%) predominated among the viruses detected, followed, with lesser prevalence, by porcine circovirus 2 (40 of 154; 26%) and swine influenza A virus (19 of 154; 12%). Bacterial coinfection and coinfection of bacteria and viruses were common (128 of 154; 83%). A strong positive correlation was found between coinfection by H. parasuis and M. hyorhinis and also by H. parasuis with PRRSV.

Anisoplanatic adaptive optics in parallelized laser scanning microscopy.

Inhomogeneities in the refractive index of a biological microscopy sample can introduce phase aberrations, severely impairing the quality of images. Adaptive optics can be employed to correct for phase aberrations and improve image quality. However, conventional adaptive optics can only correct a single phase aberration for the whole field of view (isoplanatic correction) while, due to the highly heterogeneous nature of biological tissues, the sample induced aberrations in microscopy often vary throughout the field of view (anisoplanatic aberration), limiting significantly the effectiveness of adaptive optics. This paper reports on a new approach for aberration correction in laser scanning confocal microscopy, in which a spatial light modulator is used to generate multiple excitation points in the sample to simultaneously scan different portions of the field of view with completely independent correction, achieving anisoplanatic compensation of sample induced aberrations, in a significantly shorter time compared to sequential isoplanatic correction of multiple image subregions. The method was tested in whole Drosophila brains and in larval Zebrafish, each showing a dramatic improvement in resolution and sharpness when compared to conventional isoplanatic adaptive optics.

Expanding clinical spectrum of Caspr2 antibody-associated disease: warning on brainstem involvement and respiratory failure.

We report the case of a 68-year-old man who presented with ataxia, insomnia, rapidly developing cognitive decline, seizures and small vessel vasculitis. Both serum and cerebro-spinal fluid samples showed positive titre of anti-CASPR2 antibodies. Limbic encephalitis was diagnosed and immunomodulatory therapy was started with benefit. After one-year follow-up, the patient relapsed with a difficult-to-treat respiratory failure, brainstem involvement, neuropathic pain and severe dysautonomia with esophageal dysfunction. We discuss here the occurrence of life-threating complication such as respiratory dysfunction in CASPR2 limbic encephalitis. Furthermore, we showed different phenotype and treatment response during disease onset compared to relapse. This case expands the clinical spectrum of anti-CASPR2 associated disease, underlying the need for respiratory and sleep evaluation.

New electromechanical substrate abnormalities in high-risk patients with Brugada syndrome.

BACKGROUND: The relationship between the typical electrocardiographic pattern and electromechanical abnormalities has never been systematically explored in Brugada syndrome (BrS). OBJECTIVES: The aims of this study were to characterize the electromechanical substrate in patients with BrS and to evaluate the relationship between electrical and mechanical abnormalities. METHODS: We enrolled 50 consecutive high-risk patients with BrS (mean age 42 ± 7.2 years), with implantable cardioverter-defibrillator implantation for primary or secondary prevention of ventricular tachyarrhythmias (ventricular tachycardia/ventricular fibrillation [VT/VF]), undergoing substrate mapping and ablation. Patients underwent 3-dimensional (3D) echocardiography with 3D wall motion/deformation quantification and electroanatomic mapping before and after ajmaline administration (1 mg/kg in 5 minutes); 3D mechanical changes were compared with 50 age- and sex-matched controls. The effect of substrate ablation on electromechanical abnormalities was also assessed. RESULTS: In all patients, ajmaline administration induced Brugada type 1 pattern, with a significant increase in the electrical substrate (P < .001), particularly in patients with previous spontaneous VT/VF (P = .007). Induction of Brugada pattern was associated with lowering of right ventricular (RV) ejection fraction (P < .001) and worsening of 3D RV mechanical function (P < .001), particularly in the anterior free wall of the RV outflow tract, without changes in controls. RV electrical and mechanical abnormalities were highly correlated (r = 0.728, P < .001). By multivariate analysis, only the area of RV dysfunction was an independent predictor of spontaneous VT/VF (odds ratio 1.480; 95% confidence interval 1.159-1.889; P = .002). Substrate ablation abolished both BrS-electrocardiographic pattern and mechanical abnormalities, despite ajmaline rechallenge. CONCLUSION: BrS is an electromechanical disease affecting the RV. The typical BrS pattern reflects an extensive RV arrhythmic substrate, driving consistent RV mechanical abnormalities. Substrate ablation abolished both Brugada pattern and mechanical abnormalities.

Novel SCN5A Frameshift Mutation in Brugada Syndrome Associated With Complex Arrhythmic Phenotype.

In this case report, we characterize a novel inherited frameshift mutation c.4700_4701del (p.Phe1567Cysfs*221) in a single copy of the SCN5A gene and its association with Brugada syndrome (BrS). The proband experienced a life-threatening ventricular arrhythmia successfully treated with DC-shock and he also suffered from supraventricular tachycardia. Ajmaline test confirmed the BrS diagnosis. No other mutation nor low frequency variants in the other 23 analyzed genes were detected. The same mutation was found in the father and sister, who were both diagnosed with BrS. We hypothesize that this mutation could be responsible for BrS and potentially linked to supraventricular tachycardias. Further studies are needed to confirm this observation and to assess the clinical relevance of this mutation, in terms of risk-stratification.

Pupil mask diversity for image correction in microscopy.

Three-dimensional microscopy suffers from sample-induced aberrations that reduce the resolution and lead to misinterpretations of the object distribution. In this paper, the resolution of a three-dimensional fluorescent microscope is significantly improved by introducing an amplitude diversity in the form of a binary amplitude mask positioned in several different orientations within the pupil, followed by computer processing of the diversity images. The method has proved to be fast, easy to implement, and cost-effective in high-resolution imaging of casper fli:GFP zebrafish.

General Anesthesia Attenuates Brugada Syndrome Phenotype Expression: Clinical Implications From a Prospective Clinical Trial.

OBJECTIVES: This study investigates the electrocardiographic-electrophysiological effects of administration of anesthetic drugs for general anesthesia (GA) in patients with BrS at high risk of sudden cardiac death (SCD). BACKGROUND: The safety of anesthetic agents in Brugada syndrome (BrS) is under debate. METHODS: All consecutive patients with spontaneous type 1 BrS electrocardiographic (ECG) patterns undergoing epicardial ablation of the arrhythmogenic substrate (AS) under GA were enrolled. Anesthesia was induced with single bolus of propofol and maintained with sevofluorane. ECG measurements were collected before, immediately after, and 20 min after induction of GA. Three-dimensional maps during GA and after ajmaline indicated the epicardial AS before ablation. RESULTS: Thirty-six patients with BrS (32 male, 88.9%; mean age 38.8 ± 12.0 years) with a spontaneous type 1 ECG pattern underwent GA. Induction was performed using propofol at mean dose of 1.6 to 2.6 mg/kg (2.1 ± 0.3 mg/kg). Twenty-eight (28 of 36, 77.8%) patients showed a reversion to a nondiagnostic pattern. ST-segment elevation (0.32 ± 0.01 mV vs. 0.19 ± 0.02 mV; p < 0.001) and J-wave amplitude (0.47 ± 0.02 mV vs. 0.31 ± 0.03 mV; p < 0.001) decreased after propofol. The AS area during GA, in the absence of BrS pattern, significantly enlarged after administration of ajmaline (3.6 ± 0.5 cm2 vs. 20.3 ± 0.8 cm2). No patient developed malignant arrhythmias during GA induction and maintenance. CONCLUSIONS: This study shows that GA using single-bolus propofol and volatile anesthetics is safe in high-risk patients with BrS, and it may exert a modulating effect by reducing the manifestation of type 1 BrS pattern and AS in the form of epicardial abnormal ECGs. (Epicardial Ablation in Brugada Syndrome: An Extension Study of 200 BrS Patients; NCT03106701).

Assessing the Malignant Ventricular Arrhythmic Substrate in Patients With Brugada Syndrome.

BACKGROUND: Guidelines recommend the use of implanted cardioverter-defibrillators in patients with Brugada syndrome and induced ventricular tachyarrhythmias, but there is no evidence supporting it. OBJECTIVES: This prospective registry study was designed to explore clinical and electrophysiological predictors of malignant ventricular tachyarrhythmia inducibility in Brugada syndrome. METHODS: A total of 191 consecutive selected patients with (group 1; n = 88) and without (group 2; n = 103) Brugada syndrome-related symptoms were prospectively enrolled in the registry. Patients underwent electrophysiological study and substrate mapping or ablation before and after ajmaline testing (1 mg/kg/5 min). RESULTS: Overall, before ajmaline testing, 53.4% of patients had ventricular tachyarrhythmia inducibility, which was more frequent in group 1 (65.9%) than in group 2 (42.7%; p < 0.001). Regardless of clinical presentation, larger substrates with more fragmented long-duration ventricular potentials were found in patients with inducible arrhythmias than in patients without inducible arrhythmias (p < 0.001). One extrastimulus was used in more extensive substrates (median 13 cm2; p < 0.001), and ventricular fibrillation was the more frequently induced rhythm (p < 0.001). After ajmaline, patients without arrhythmia inducibility had arrhythmia inducibility without a difference in substrate characteristics between the 2 groups. The substrate size was the only independent predictor of inducibility (odds ratio: 4.51; 95% confidence interval: 2.51 to 8.09; p < 0.001). A substrate size of 4 cm2 best identified patients with inducible arrhythmias (area under the curve: 0.98; p < 0.001). Substrate ablation prevented ventricular tachyarrhythmia reinducibility. CONCLUSIONS: In Brugada syndrome dynamic substrate variability represents the pathophysiological basis of lethal ventricular tachyarrhythmias. Substrate size is independently associated with arrhythmia inducibility, and its determination after ajmaline identifies high-risk patients missed by clinical criteria. Substrate ablation is associated with electrocardiogram normalization and not arrhythmia reinducibility. (Epicardial Ablation in Brugada Syndrome [BRUGADA_I]; NCT02641431; Epicardial Ablation in Brugada Syndrome: An Extension Study of 200 BrS Patients; NCT03106701).

Optimal model-based sensorless adaptive optics for epifluorescence microscopy.

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.