Germicidal efficacy of continuous and pulsed ultraviolet-C radiation on pathogen models and SARS-CoV-2.

Ultraviolet radiation's germicidal efficacy depends on several parameters, including wavelength, radiant exposure, microbial physiology, biological matrices, and surfaces. In this work, several ultraviolet radiation sources (a low-pressure mercury lamp, a KrCl excimer, and four UV LEDs) emitting continuous or pulsed irradiation were compared. The greatest log reductions in E. coli cells and B. subtilis endospores were 4.1 ± 0.2 (18 mJ cm-2) and 4.5 ± 0.1 (42 mJ cm-2) with continuous 222 nm, respectively. The highest MS2 log reduction observed was 2.7 ± 0.1 (277 nm at 3809 mJ cm-2). Log reductions of SARS-CoV-2 with continuous 222 nm and 277 nm were ≥ 3.4 ± 0.7, with 13.3 mJ cm-2 and 60 mJ cm-2, respectively. There was no statistical difference between continuous and pulsed irradiation (0.83-16.7% [222 nm and 277 nm] or 0.83-20% [280 nm] duty rates) on E. coli inactivation. Pulsed 260 nm radiation (0.5% duty rate) at 260 nm yielded significantly greater log reduction for both bacteria than continuous 260 nm radiation. There was no statistical difference in SARS-CoV-2 inactivation between continuous and pulsed 222 nm UV-C radiation and pulsed 277 nm radiation demonstrated greater germicidal efficacy than continuous 277 nm radiation. Greater radiant exposure for all radiation sources was required to inactivate MS2 bacteriophage. Findings demonstrate that pulsed irradiation could be more useful than continuous UV radiation in human-occupied spaces, but threshold limit values should be respected. Pathogen-specific sensitivities, experimental setup, and quantification methods for determining germicidal efficacy remain important factors when optimizing ultraviolet radiation for surface decontamination or other applications.

Strategies for uncovering stable isotope tracing patterns between cell populations.

Despite practical complexities, isotope tracing studies in humans are becoming increasingly feasible. However, several technological challenges need to be addressed in order to take full advantage of human tracing studies. First, absolute metabolic flux measurements in mice are not so easily applied to human models, given that tissue resection is restricted to a single surgical time point. Second, isotope tracing has yet to be employed to detect metabolic differences between cells types in vivo. Here, we discuss the current models and propose an alternative, liquid tumor environment, that could overcome these limitations. Furthermore, we highlight current strategies used to maintain isotopolog enrichment following cell isolation techniques to facilitate cell-type-specific analysis.

Assessing the Immune Regulatory Role of Metabolites In Vitro.

One method of immune evasion that cancer cells employ is the secretion of immune regulatory metabolites into the tumor microenvironment (TME). These metabolites can promote immunosuppressive cell subsets, while inhibiting key tumor-killing subsets, such as T cells. Thus, the identification of these metabolites may help develop methods for improving cell-based therapy. However, after identifying a potential immune regulatory metabolite, it is crucial to assess the impacts of the metabolite on T cell immunobiology. In this chapter, we describe an in vitro method of testing and analyzing the influence of a specific metabolite on T cell proliferation and function.

Principles of reproducible metabolite profiling of enriched lymphocytes in tumors and ascites from human ovarian cancer.

Identifying metabolites and delineating their immune-regulatory contribution in the tumor microenvironment is an area of intense study. Interrogating metabolites and metabolic networks among immune cell subsets and host cells from resected tissues and fluids of human patients presents a major challenge, owing to the specialized handling of samples for downstream metabolomics. To address this, we first outline the importance of collaborating with a biobank for coordinating and streamlining workflow for point of care, sample collection, processing and cryopreservation. After specimen collection, we describe our 60-min rapid bead-based cellular enrichment method that supports metabolite analysis between T cells and tumor cells by mass spectrometry. We also describe how the metabolic data can be complemented with metabolic profiling by flow cytometry. This protocol can serve as a foundation for interrogating the metabolism of cell subsets from primary human ovarian cancer.

Clinically relevant T cell expansion media activate distinct metabolic programs uncoupled from cellular function.

Ex vivo expansion conditions used to generate T cells for immunotherapy are thought to adopt metabolic phenotypes that impede therapeutic efficacy in vivo. The comparison of five different culture media used for clinical T cell expansion revealed unique optima based on different output variables, including proliferation, differentiation, function, activation, and mitochondrial phenotypes. The extent of proliferation and function depended on the culture media rather than stimulation conditions. Moreover, the expanded T cell end products adapted their metabolism when switched to a different media formulation, as shown by glucose and glutamine uptake and patterns of glucose isotope labeling. However, adoption of these metabolic phenotypes was uncoupled to T cell function. Expanded T cell products cultured in ascites from ovarian cancer patients displayed suppressed mitochondrial activity and function irrespective of the ex vivo expansion media. Thus, ex vivo T cell expansion media have profound impacts on metabolism and function.

1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer.

Immune regulatory metabolites are key features of the tumor microenvironment (TME), yet with a few exceptions, their identities remain largely unknown. Here, we profiled tumor and T cells from tumor and ascites of patients with high-grade serous carcinoma (HGSC) to uncover the metabolomes of these distinct TME compartments. Cells within the ascites and tumor had pervasive metabolite differences, with a notable enrichment in 1-methylnicotinamide (MNA) in T cells infiltrating the tumor compared with ascites. Despite the elevated levels of MNA in T cells, the expression of nicotinamide N-methyltransferase, the enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine to nicotinamide, was restricted to fibroblasts and tumor cells. Functionally, MNA induces T cells to secrete the tumor-promoting cytokine tumor necrosis factor alpha. Thus, TME-derived MNA contributes to the immune modulation of T cells and represents a potential immunotherapy target to treat human cancer.

Cognitive Reserve Proxies Do Not Differentially Account for Cognitive Performance in Patients with Focal Frontal and Non-Frontal Lesions.

OBJECTIVE: Cognitive reserve (CR) suggests that premorbid efficacy, aptitude, and flexibility of cognitive processing can aid the brain's ability to cope with change or damage. Our previous work has shown that age and literacy attainment predict the cognitive performance of frontal patients on frontal-executive tests. However, it remains unknown whether CR also predicts the cognitive performance of non-frontal patients. METHOD: We investigated the independent effect of a CR proxy, National Adult Reading Test (NART) IQ, as well as age and lesion group (frontal vs. non-frontal) on measures of executive function, intelligence, processing speed, and naming in 166 patients with focal, unilateral frontal lesions; 91 patients with focal, unilateral non-frontal lesions; and 136 healthy controls. RESULTS: Fitting multiple linear regression models for each cognitive measure revealed that NART IQ predicted executive, intelligence, and naming performance. Age also significantly predicted performance on the executive and processing speed tests. Finally, belonging to the frontal group predicted executive and naming performance, while membership of the non-frontal group predicted intelligence. CONCLUSIONS: These findings suggest that age, lesion group, and literacy attainment play independent roles in predicting cognitive performance following stroke or brain tumour. However, the relationship between CR and focal brain damage does not differ in the context of frontal and non-frontal lesions.

Understanding lymphocyte metabolism for use in cancer immunotherapy.

Like all dividing cells, naïve T cells undergo a predictable sequence of events to enter the cell cycle starting from G0 and progressing to G1 , S and finally G2 /M. This methodical series of steps ensures fidelity in the generation of two identical T cells during a single round of division. To achieve this, T cells must activate or inactivate metabolic pathways at discrete times during each phase of the cell cycle. This permits the generation of substrates to support biosynthesis, bioenergetics and the epigenetic changes required for proper differentiation and function. The precursors that feed into these pathways are often shared, highlighting the complex relationship between metabolism and cellular processes that are essential to lymphocytes. It is therefore not surprising that different T cell subtypes exhibit unique metabolic dependencies that change as they mature and go through specialized differentiation programmes. The importance of the influence of metabolism on T cells is underscored by the emerging field of cancer immunotherapy, where autologous T cells can be manufactured ex vivo then infused as a form of curative treatment for human cancers. This review will highlight some of the recent knowledge on T lymphocyte metabolism and give a perspective on the practical implications for cellular-based immunotherapy.

Ex Vivo Detection of Circulating Tumor Cells from Whole Blood by Direct Nanoparticle Visualization.

The detection of circulating tumor cells (CTCs) from blood samples can predict prognosis, response to systemic chemotherapy, and metastatic spread of carcinoma. Therefore, approaches for CTC identification is an important aspect of current cancer research. Here, a method for the direct visualization of nanoparticle-coated CTCs under dark field illumination is presented. A metastatic breast cancer cell line (4T1) was transduced with a non-native target protein (Thy1.1). Positive 4T1-Thy1.1 cells incubated with antibody-coated metallic nanoshells appeared overly bright at low magnification, allowing a quick screening of samples and easy visual detection of even single isolated CTCs. The use of a nontransduced cell line as control creates the ideal scenario to evaluate nonspecific binding. A murine metastatic tumor model with the 4T1-Thy1.1 cell line was also implemented. Blood was drawn from mice over the course of one month, and CTCs were successfully detected in all positive subjects. This work validates the use of metallic nanoshells as labels for direct visualization of CTCs while providing guidelines to a systematic development of nanotechnology-based detection systems for CTCs.

Clinical Evaluation of Silent T1-Weighted MRI and Silent MR Angiography of the Brain.

OBJECTIVE: New MRI sequences based on rapid radial acquisition have reduced gradient noise. The purpose of this study was to compare Silent T1-weighted and unenhanced MR angiography (MRA) against conventional sequences in a clinical population. MATERIALS AND METHODS: The study cohort consisted of 40 patients with suspected brain metastases (median age, 60 years; range, 23-91 years) who underwent T1-weighted contrast-enhanced MRI and 51 patients with suspected vascular lesions or cerebral ischemia (median age, 60 years; range, 16-94 years) who underwent unenhanced intracranial MRA. Three neuroradiologists reviewed the images blindly and rated several measures of image quality on a 5-point Likert scale. Reviewers recorded the number of enhancing lesions and whether Silent images were better than, worse than, or equivalent to conventional images. RESULTS: For T1-weighted MR images, ratings were slightly lower for Silent versus conventional images, except for diagnostic confidence. Although more lesions were detected on conventional images, this difference was not statistically significant; agreement was seen in 88% of cases. In 48% of cases, T1-weighted scans were deemed equivalent, but when a preference existed, it was usually for conventional images (38% vs 14%). Conventional MRA images were rated higher on all image quality metrics and were strongly preferred (reviewers preferred conventional images in 69% of cases, rated the images as equivalent in 27% of cases, and preferred Silent images in 4% of cases). In some cases, artifacts on Silent images caused reduced vessel caliber, vessel irregularities, and even absent vessels. CONCLUSION: Although conventional T1-weighted images were preferred overall, most Silent T1-weighted images were rated as equivalent to or better than conventional images and represent a potential alternative for imaging of noise-averse patients. Silent MRA scored significantly worse and could not be recommended at this time, suggesting that it requires additional refinement before routine clinical use.

The Doors and People Test: The effect of frontal lobe lesions on recall and recognition memory performance.

OBJECTIVE: Memory deficits in patients with frontal lobe lesions are most apparent on free recall tasks that require the selection, initiation, and implementation of retrieval strategies. The effect of frontal lesions on recognition memory performance is less clear with some studies reporting recognition memory impairments but others not. The majority of these studies do not directly compare recall and recognition within the same group of frontal patients, assessing only recall or recognition memory performance. Other studies that do compare recall and recognition in the same frontal group do not consider recall or recognition tests that are comparable for difficulty. Recognition memory impairments may not be reported because recognition memory tasks are less demanding. METHOD: This study aimed to investigate recall and recognition impairments in the same group of 47 frontal patients and 78 healthy controls. The Doors and People Test was administered as a neuropsychological test of memory as it assesses both verbal and visual recall and recognition using subtests that are matched for difficulty. RESULTS: Significant verbal and visual recall and recognition impairments were found in the frontal patients. CONCLUSION: These results demonstrate that when frontal patients are assessed on recall and recognition memory tests of comparable difficulty, memory impairments are found on both types of episodic memory test.

The effect of age on cognitive performance of frontal patients.

Age is known to affect prefrontal brain structure and executive functioning in healthy older adults, patients with neurodegenerative conditions and TBI. Yet, no studies appear to have systematically investigated the effect of age on cognitive performance in patients with focal lesions. We investigated the effect of age on the cognitive performance of a large sample of tumour and stroke patients with focal unilateral, frontal (n=68), or non-frontal lesions (n=45) and healthy controls (n=52). We retrospectively reviewed their cross sectional cognitive and imaging data. In our frontal patients, age significantly predicted the magnitude of their impairment on two executive tests (Raven's Advanced Progressive Matrices, RAPM and the Stroop test) but not on nominal (Graded Naming Test, GNT) or perceptual (Incomplete Letters) task. In our non-frontal patients, age did not predict the magnitude of their impairment on the RAPM and GNT. Furthermore, the exacerbated executive impairment observed in our frontal patients manifested itself from middle age. We found that only age consistently predicted the exacerbated executive impairment. Lesions to specific frontal areas, or an increase in global brain atrophy or white matter abnormalities were not associated with this impairment. Our results are in line with the notion that the frontal cortex plays a critical role in aging to counteract cognitive and neuronal decline. We suggest that the combined effect of aging and frontal lesions impairs the frontal cortical systems by causing its computational power to fall below the threshold needed to complete executive tasks successfully.

Survival of effector CD8+ T cells during influenza infection is dependent on autophagy.

The activation and expansion of effector CD8(+) T cells are essential for controlling viral infections and tumor surveillance. During an immune response, T cells encounter extrinsic and intrinsic factors, including oxidative stress, nutrient availability, and inflammation, that can modulate their capacity to activate, proliferate, and survive. The dependency of T cells on autophagy for in vitro and in vivo activation, expansion, and memory remains unclear. Moreover, the specific signals and mechanisms that activate autophagy in T effector cells and their survival are not known. In this study, we generated a novel inducible autophagy knockout mouse to study T cell effector responses during the course of a virus infection. In response to influenza infection, Atg5(-/-) CD8(+) T cells had a decreased capacity to reach the peak effector response and were unable to maintain cell viability during the effector phase. As a consequence of Atg5 deletion and the impairment in effector-to-memory cell survival, mice fail to mount a memory response following a secondary challenge. We found that Atg5(-/-) effector CD8(+) T cells upregulated p53, a transcriptional state that was concomitant with widespread hypoxia in lymphoid tissues of infected mice. The onset of p53 activation was concurrent with higher levels of reactive oxygen species (ROS) that resulted in ROS-dependent apoptotic cell death, a fate that could be rescued by treating with the ROS scavenger N-acetylcysteine. Collectively, these results demonstrate that effector CD8(+) T cells require autophagy to suppress cell death and maintain survival in response to a viral infection.

Tumor vascularity in ovarian cancer: T cells need breathing room.

Tumor-infiltrating lymphocytes (TILs) are crucial for effective antitumor responses. However, hypoxia can skew T-cell differentiation and function, thereby perturbing TILs. We have demonstrated that TILs and their immune function are associated with tumor vascularization. These features are prognostic for improved disease-specific survival in ovarian cancer. Thus, new immunotherapies should consider how hypoxia impacts antitumor immunity.

White matter integrity assessed by diffusion tensor tractography in a patient with a large tumor mass but minimal clinical and neuropsychological deficits.

Diffusion tensor imaging (DTI) tractography and image registration were used to investigate a patient with a massive left-sided brain tumor, whose size was largely disproportionate to his subtle neurological deficits. MRI was obtained from the patient and his healthy identical twin, who acted as anatomical reference for DTI and as a control for quantitative measures. To compensate for the patient's altered anatomy, seed and way points for probabilistic tractography were drawn on the color-coded direction maps of the healthy twin. Registration, based on the combination of b0-images, T2-weighted and T1-weighted images, was used to identify the corresponding regions in the patient. The corticospinal tract (CST), the superior longitudinal fasciculus (SLF), and the cingulum bundle (CB) showed displaced anatomy. A significant difference was found between fractional anisotropy distribution along the left SLF and CB, but not along the CST. These findings fit well with the patient's substantial preservation of his motor abilities, while abnormalities of the SLF and CB could explain the subtle but detectable cognitive deficits.

Effect of frontal lobe lesions on the recollection and familiarity components of recognition memory.

Single-process theories assume that familiarity is the sole influence on recognition memory with decisions being made as a continuous process. Dual-process theories claim that recognition involves both recollection and familiarity processes with recollection as a threshold process. Although, the frontal lobes of the brain play an important role in recognition memory, few studies have examined the effect of frontal lobe lesions on recollection and familiarity. In the current study, the nonverbal recognition memory of 24 patients with focal frontal lesions due to tumour or stroke was examined. Recollection and familiarity were estimated using the receiver operating characteristic (ROC) method. A secondary analysis was also conducted using standard signal detection theory methodology. Both analyses led to similar conclusions where only the familiarity component of recognition memory was impaired in frontal patients compared to healthy controls whilst the recollection-type (or variance ratio) processes remained intact.

A fungal family of transcriptional regulators: the zinc cluster proteins.

The trace element zinc is required for proper functioning of a large number of proteins, including various enzymes. However, most zinc-containing proteins are transcription factors capable of binding DNA and are named zinc finger proteins. They form one of the largest families of transcriptional regulators and are categorized into various classes according to zinc-binding motifs. This review focuses on one class of zinc finger proteins called zinc cluster (or binuclear) proteins. Members of this family are exclusively fungal and possess the well-conserved motif CysX(2)CysX(6)CysX(5-12)CysX(2)CysX(6-8)Cys. The cysteine residues bind to two zinc atoms, which coordinate folding of the domain involved in DNA recognition. The first- and best-studied zinc cluster protein is Gal4p, a transcriptional activator of genes involved in the catabolism of galactose in the budding yeast Saccharomyces cerevisiae. Since the discovery of Gal4p, many other zinc cluster proteins have been characterized; they function in a wide range of processes, including primary and secondary metabolism and meiosis. Other roles include regulation of genes involved in the stress response as well as pleiotropic drug resistance, as demonstrated in budding yeast and in human fungal pathogens. With the number of characterized zinc cluster proteins growing rapidly, it is becoming more and more apparent that they are important regulators of fungal physiology.