Diffusion Mapping with Diffractive Optical Elements for Periodically Patterned Photobleaching.

Fourier transform-fluorescence recovery after photobleaching (FT-FRAP) using a diffractive optical element (DOE) is shown to support distance-dependent diffusion analysis in biologically relevant media. Integration of DOEs enables patterning of a dot array for parallel acquisition of point-bleach FRAP measurements at multiple locations across the field of view. In homogeneous media, the spatial harmonics of the dot array analyzed in the spatial Fourier transform domain yield diffusion recovery curves evaluated over specific well-defined distances. Relative distances for diffusive recovery in the spatial Fourier transform domain are directly connected to the 2D (h,k) Miller indices of the corresponding lattice lines. The distribution of the photobleach power across the entire field of view using a multidot array pattern greatly increases the overall signal power in the spatial FT-domain for signal-to-noise improvements. Derivations are presented for the mathematical underpinnings of FT-FRAP performed with 2D periodicity in the photobleach patterns. Retrofitting of FT-FRAP into instrumentation for high-throughput FRAP analysis (Formulatrix) supports automated analysis of robotically prepared 96-well plates for precise quantification of molecular mobility. Figures of merit are evaluated for FT-FRAP in analysis for both slow diffusion of fluorescent dyes in glassy polymer matrices spanning several days and model proteins and monoclonal antibodies within aqueous solutions recovering in matters of seconds.

Periodic Photobleaching with Structured Illumination for Diffusion Imaging.

The use of periodically structured illumination coupled with spatial Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) was shown to support diffusivity mapping within segmented domains of arbitrary shape. Periodic "comb-bleach" patterning of the excitation beam during photobleaching encoded spatial maps of diffusion onto harmonic peaks in the spatial Fourier transform. Diffusion manifests as a simple exponential decay of a given harmonic, improving the signal to noise ratio and simplifying mathematical analysis. Image segmentation prior to Fourier transformation was shown to support pooling for signal to noise enhancement for regions of arbitrary shape expected to exhibit similar diffusivity within a domain. Following proof-of-concept analyses based on simulations with known ground-truth maps, diffusion imaging by FT-FRAP was used to map spatially-resolved diffusion differences within phase-separated domains of model amorphous solid dispersion spin-cast thin films. Notably, multi-harmonic analysis by FT-FRAP was able to definitively discriminate and quantify the roles of internal diffusion and exchange to higher mobility interfacial layers in modeling the recovery kinetics within thin amorphous/amorphous phase-separated domains, with interfacial diffusion playing a critical role in recovery. These results have direct implications for the design of amorphous systems for stable storage and efficacious delivery of therapeutic molecules.

Long-Term Efficacy and Safety of Fecal Microbiota Transplantation for C. difficile Infections Across Academic and Private Clinical Settings.

PURPOSE: Fecal microbiota transplant (FMT) is increasingly performed for Clostridioides difficile infection (CDI), although long-term efficacy and safety data are limited and are focused on results from academic medical centers rather than private settings where most patients receive care. METHODS: Medical records of 165 patients who received FMTs for CDI were reviewed from an academic medical center and an adjacent, unaffiliated private practice. Of these patients, 68 also completed a survey regarding their long-term disease course and interval health. RESULTS: CDI resolution occurred in 81.3% (100/123) at the academic center and 95.2% (40/42) in the private setting. Private practice patients were more likely to present with recurrent, rather than refractory, CDI (92.9% vs. 66.7% P <0.001). Those from the academic center were more likely to have comorbid IBD, recent hospitalization, recent proton pump inhibitor use, ongoing immunosuppression, and inpatient FMT (all P values <0.05).Among surveyed patients, 29.4% developed interval comorbidities or changes to pre-existing conditions after a median follow-up of 33.7 months (IQR 13.2 to 44.3 mo). Of 30 patients requiring subsequent antibiotics, 13.3% suffered CDI relapse. All subjects who had initially responded to FMT but had a subsequent CDI (17.9%, 10/56) responded to another FMT. CONCLUSIONS: In a real-world setting, patients who underwent FMT at academic centers differed significantly in clinical characteristics from those treated at a private practice. In both settings, FMT is an effective treatment for CDI not responding to standard therapies, even after subsequent antibiotic use. New diagnoses following FMT, however, are common and merit further exploration.

Stochastic Differential Scanning Calorimetry by Nonlinear Optical Microscopy.

Stochastic phase transformations within individual crystalline particles were recorded by integration of second harmonic generation (SHG) imaging with differential scanning calorimetry (DSC). The SHG activity of a crystal is highly sensitive to the specific molecular packing arrangement within a noncentrosymmetric lattice, providing access to information otherwise unavailable by conventional imaging approaches. Consequently, lattice transformations associated with dehydration/desolvation events were readily observed by SHG imaging and directly correlated to the phase transformations detected by the DSC measurements. Following studies of a model system (urea), stochastic differential scanning calorimetry (SDSC) was performed on trehalose dihydrate, which has a more complex phase behavior. From these measurements, SDSC revealed a broad diversity of single-particle thermal trajectories and direct evidence of a "cold phase transformation" process not observable by the DSC measurements alone.

Spectral classification by generative adversarial linear discriminant analysis.

Generative adversarial linear discriminant analysis (GALDA) is formulated as a broadly applicable tool for increasing classification accuracy and reducing overfitting in spectrochemical analysis. Although inspired by the successes of generative adversarial neural networks (GANs) for minimizing overfitting artifacts in artificial neural networks, GALDA was built around an independent linear algebra framework distinct from those in GANs. In contrast to feature extraction and data reduction approaches for minimizing overfitting, GALDA performs data augmentation by identifying and adversarially excluding the regions in spectral space in which genuine data do not reside. Relative to non-adversarial analogs, loading plots for dimension reduction showed significant smoothing and more prominent features aligned with spectral peaks following generative adversarial optimization. Classification accuracy was evaluated for GALDA together with other commonly available supervised and unsupervised methods for dimension reduction in simulated spectra generated using an open-source Raman database (Romanian Database of Raman Spectroscopy, RDRS). Spectral analysis was then performed for microscopy measurements of microsphereroids of the blood thinner clopidogrel bisulfate and in THz Raman imaging of common constituents in aspirin tablets. From these collective results, the potential scope of use for GALDA is critically evaluated relative to alternative established spectral dimension reduction and classification methods.

Proposed BioRepository platform solution for the ALS research community.

ALS is a rare disorder whose cause and pathogenesis is largely unknown ( 1 ). There is a recognized need to develop biomarkers for ALS to better understand the disease, expedite diagnosis and to facilitate therapy development. Collaboration is essential to obtain a sufficient number of samples to allow statistically meaningful studies. The availability of high quality biological specimens for research purposes requires the development of standardized methods for collection, long-term storage, retrieval and distribution of specimens. The value of biological samples to scientists and clinicians correlates with the completeness and relevance of phenotypical and clinical information associated with the samples ( 2 , 3 ). While developing a secure Web-based system to manage an inventory of multi-site BioRepositories, algorithms were implemented to facilitate ad hoc parametric searches across heterogeneous data sources that contain data from clinical trials and research studies. A flexible schema for a barcode label was introduced to allow association of samples to these data. The ALSBank™ BioRepository platform solution for managing biological samples and associated data is currently deployed by the Northeast ALS Consortium (NEALS). The NEALS Consortium and the Massachusetts General Hospital (MGH) Neurology Clinical Trials Unit (NCTU) support a network of multiple BioBanks, thus allowing researchers to take advantage of a larger specimen collection than they might have at an individual institution. Standard operating procedures are utilized at all collection sites to promote common practices for biological sample integrity, quality control and associated clinical data. Utilizing this platform, we have created one of the largest virtual collections of ALS-related specimens available to investigators studying ALS.

Faecal microbiota transplantation for diarrhoea-predominant irritable bowel syndrome: a double-blind, randomised, placebo-controlled trial.

BACKGROUND: Faecal microbiota transplantation (FMT) has shown promise in alleviating the symptoms of irritable bowel syndrome (IBS); however, controlled data on this technique are scarce. The aim of this clinical trial was to assess the efficacy of FMT in alleviating diarrhoea-predominant IBS (IBS-D). METHODS: We did a double-blind, randomised, placebo-controlled crossover trial in patients aged 18-65 years with moderate-to-severe IBS-D defined by an IBS-Symptom Severity Score (IBS-SSS) of more than 175, recruited from three US centres. Patients were randomly assigned (1:1) in blocks of four with a computer-generated randomisation sequence to receive FMT capsules followed by identical-appearing placebo capsules, or placebo capsules followed by FMT capsules. All participants and study team members were masked to randomisation. An independent staff member assigned the treatments according to consecutive numbers. Patients received either 75 FMT capsules (each capsule contained approximately 0·38 g of minimally processed donor stool) or 75 placebo capsules over 3 days (25 capsules per day). All patients crossed over to the alternate treatment at 12 weeks. The primary outcome was difference in IBS-SSS between the groups at 12 weeks. Intention-to-treat analyses were done and all patients who received study drug were included in an adverse events analysis. The trial was terminated during recruitment because results from an interim analysis revealed futility. The study is registered with ClinicalTrials.gov, number NCT02328547. FINDINGS: From May 28, 2015, to April 21, 2017, 48 patients were randomly assigned to receive FMT first (n=25) or placebo first (n=23). Three participants were lost to follow-up in the FMT group. IBS-SSS did not differ between FMT recipients (mean 221 [SD 105]) and placebo recipients (236 [95]) at 12 weeks (p=0·65), after adjustment for baseline scores. The most common drug-related adverse events included abdominal pain (five [10%] of the 48 participants while receiving FMT capsules vs four [8%] while receiving placebo), nausea (four [8%] vs two [4%]), and exacerbation of diarrhoea (three [6%] vs eight [17%]). One serious adverse event that was unrelated to study drug (acute cholecystitis) was reported in a patient while receiving placebo capsules. INTERPRETATION: FMT was safe, but did not induce symptom relief at 12 weeks compared with placebo. Additional studies are needed to determine the efficacy of FMT for IBS-D. FUNDING: National Institutes of Health.

How common are ALS plateaus and reversals?

OBJECTIVE: To determine the frequency of amyotrophic lateral sclerosis (ALS) plateaus and reversals in the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) database. METHODS: We analyzed Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS) and ALSFRS-revised (ALSFRS-R) data from PRO-ACT participants. The frequencies of participants experiencing plateaus (periods where scores did not change) were calculated over 6-, 12-, and 18-month epochs. The percentage of participants ever experiencing reversals (periods where scores improved) of different lengths were also calculated and plotted. RESULTS: Over 6 months, 25% of 3,132 participants did not decline. Over 12 months, 16% of 2,105 participants did not decline. Over 18 months, 7% of 1,218 participants did not decline. Small ALS reversals were also common, especially over shorter follow-up intervals; 14% of 1,343 participants had a 180-day interval where their ALSFRS-R slope was greater than zero. Fewer than 1% of participants ever experienced improvements of 4 or more ALSFRS-R points lasting at least 12 months. CONCLUSION: ALS plateaus and small reversals are common, especially over brief intervals. In light of these data, stable disease, especially for a short period of time, should not be interpreted as an ALS treatment effect. Large sustained ALS reversals, on the other hand, are rare, potentially important, and warrant further study.

Endoscopy plays an important preoperative role in bariatric surgery.

BACKGROUND: The role of upper endoscopy (EGD) in obese patients prior to bariatric surgery is controversial. The aim of this study was to evaluate the diagnostic yield and cost of routine EGD before bariatric surgery. METHODS: The medical records of consecutive obese patients who underwent EGD prior to bariatric surgery between May 2000 and September 2002 were reviewed. Two experienced endoscopists reviewed all EGD reports, and findings were divided into 4 groups based on predetermined criteria: group 0 (normal study), group 1 (abnormal findings that neither changed the surgical approach nor postponed surgery), group 2 (abnormal findings that changed the surgical approach or postponed surgery), and group 3 (results that were an absolute contraindication to surgery). Clinically important findings included lesions in groups 2 and 3. The cost of EGD (430.72 US dollars) was estimated using the endoscopist fee under Medicare reimbursement. RESULTS: During the 28-month study period, 195 patients were evaluated by EGD prior to bariatric surgery. One or more lesions were identified in 89.7% of patients, with 61.5% having a clinically important finding. The prevalence of endoscopic findings using the classification system above was as follows: group 0 (10.3%), group 1 (28.2%), group 2 (61.5%), and group 3 (0.0%). Overall, the most common lesions identified were hiatal hernia (40.0%), gastritis (28.7%), esophagitis (9.2%), gastric ulcer (3.6%), Barrett's esophagus (3.1%), and esophageal ulcer (3.1%). The cost of performing routine endoscopy on all patients prior to bariatric surgery was 699.92 US dollars per clinically important lesion detected. CONCLUSIONS: Routine upper endoscopy before bariatric surgery has a high diagnostic yield and has a low cost per clinically important lesion detected.

Gene profiling of human induced pluripotent stem cell-derived astrocyte progenitors following spinal cord engraftment.

The generation of human induced pluripotent stem cells (hiPSCs) represents an exciting advancement with promise for stem cell transplantation therapies as well as for neurological disease modeling. Based on the emerging roles for astrocytes in neurological disorders, we investigated whether hiPSC-derived astrocyte progenitors could be engrafted to the rodent spinal cord and how the characteristics of these cells changed between in vitro culture and after transplantation to the in vivo spinal cord environment. Our results show that human embryonic stem cell- and hiPSC-derived astrocyte progenitors survive long-term after spinal cord engraftment and differentiate to astrocytes in vivo with few cells from other lineages present. Gene profiling of the transplanted cells demonstrates the astrocyte progenitors continue to mature in vivo and upregulate a variety of astrocyte-specific genes. Given this mature astrocyte gene profile, this work highlights hiPSCs as a tool to investigate disease-related astrocyte biology using in vivo disease modeling with significant implications for human neurological diseases currently lacking animal models.

Altered astrocytic expression of TDP-43 does not influence motor neuron survival.

The role of glia as a contributing factor to motor neuron (MN) death in amyotrophic lateral sclerosis (ALS) is becoming increasingly appreciated. However, most studies implicating astrocytes have focused solely on models of ALS caused by superoxide dismutase 1 (SOD1) mutations. The goal of our study was to determine whether astrocytes contribute to wild-type MN death in the case of ALS caused by mutations in tar-DNA binding protein 43 (TDP-43). Since it is currently unknown how TDP-43 mutations cause disease, we derived astrocytes for study from both gain and loss of function mouse models of TDP-43. Astrocytes overexpressing mutant TDP-43(A315T) as well as astrocytes lacking TDP-43 were morphologically indistinguishable from wild-type astrocytes in vitro. Furthermore, astrocytes with these TDP-43 alterations did not cause the death of wild-type MNs in co-culture. To investigate the in vivo effects of TDP-43 alterations in astrocytes, glial-restricted precursors were transplanted to the wild-type rat spinal cord where they differentiated into astrocytes and interacted with host MNs. Astrocytes with TDP-43 alterations did not cause host wild-type MN damage although they were capable of engrafting and interacting with host MNs with the same efficiency as wild-type astrocytes. These data indicate that astrocytes do not adopt the same toxic phenotype as mutant SOD1 astrocytes when TDP-43 is mutated or expression levels are modified. Our study reinforces the heterogeneity in ALS disease mechanisms and highlights the potential for future screening subsets of ALS patients prior to treatment with cell type-directed therapies.

Amyotrophic lateral sclerosis: an emerging era of collaborative gene discovery.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND). It is currently incurable and treatment is largely limited to supportive care. Family history is associated with an increased risk of ALS, and many Mendelian causes have been discovered. However, most forms of the disease are not obviously familial. Recent advances in human genetics have enabled genome-wide analyses of single nucleotide polymorphisms (SNPs) that make it possible to study complex genetic contributions to human disease. Genome-wide SNP analyses require a large sample size and thus depend upon collaborative efforts to collect and manage the biological samples and corresponding data. Public availability of biological samples (such as DNA), phenotypic and genotypic data further enhances research endeavors. Here we discuss a large collaboration among academic investigators, government, and non-government organizations which has created a public repository of human DNA, immortalized cell lines, and clinical data to further gene discovery in ALS. This resource currently maintains samples and associated phenotypic data from 2332 MND subjects and 4692 controls. This resource should facilitate genetic discoveries which we anticipate will ultimately provide a better understanding of the biological mechanisms of neurodegeneration in ALS.

Fractured colon: an endoscopically distinctive lesion associated with colonic perforation following colonoscopy in patients with collagenous colitis.

BACKGROUND: Collagenous colitis is characterized by collagen deposition in the superficial colonic mucosa, beneath the surface epithelium, resulting in chronic nonbloody diarrhea of variable severity. The mucosa generally appears endoscopically normal. METHODS: We report the occurrence of distinctive linear mucosal tears, unassociated with trauma, in 4 patients during diagnostic colonoscopy. The patients' tissue specimens were examined histologically, and clinical courses were recorded. OBSERVATIONS: Recognition of linear "fractures" was followed in 3 patients by colonic perforation. One patient required colectomy. Severe collagenous colitis was present in all. The resection specimen contained shallow linear ulcers overlying fibrotic submucosa, with pneumatosis and acute peritonitis. CONCLUSIONS: We theorize that the stiffness of the colon in areas of collagenous colitis with submucosal fibrosis makes it susceptible to linear "fractures" during colonoscopic air insufflation with subsequent transmural air dissection. We urge extreme caution if this lesion is recognized at colonoscopy and recommend aborting the examination and obtaining plain radiographs to detect free intraperitoneal air.