Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow

The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2 mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12 s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120 RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400 RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5 ng, however, the optimum range determined was 2.5–10 ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR® Identifiler® Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit.     

Critical Role of STAT5 transcription factor tetramerization for cytokine responses and normal immune function

Cytokine-activated STAT proteins dimerize and bind to high-affinity motifs, and N-terminal domain-mediated oligomerization of dimers allows tetramer formation and binding to low-affinity tandem motifs, but the functions of dimers versus tetramers are unknown. We generated Stat5a-Stat5b double knockin (DKI) N-domain mutant mice in which STAT5 proteins form dimers but not tetramers, identified cytokine-regulated genes whose expression required STAT5 tetramers, and defined dimer versus tetramer consensus motifs. Whereas Stat5-deficient mice exhibited perinatal lethality, DKI mice were viable; thus, STAT5 dimers were sufficient for survival. Nevertheless, STAT5 DKI mice had fewer CD4(+)CD25(+) T cells, NK cells, and CD8(+) T cells, with impaired cytokine-induced and homeostatic proliferation of CD8(+) T cells. Moreover, DKI CD8(+) T cell proliferation after viral infection was diminished and DKI Treg cells did not efficiently control colitis. Thus, tetramerization of STAT5 is critical for cytokine responses and normal immune function, establishing a critical role for STAT5 tetramerization in vivo.     

Mean time-of-flight of photons in transillumination measurements of optically anisotropic tissue with an inclusion

We study the effect of optical anisotropy on the mean time-of-flight of photons in a slab of turbid medium containing an inclusion whose optical properties differ from those of the bulk. For this analysis the difference in the mean time for a photon introduced into the slab to reach a specified target point with and without the inclusion is calculated. This difference is defined to be a measure of the contrast. The theoretical model is based on a continuous-time random walk on a lattice, which can be solved exactly and furnishes an exact expression for the contrast. Qualitative and quantitative characteristics of the contrast are analysed as functions of the geometric configuration of the system components (locations of the source, the inclusion and the detector), parameters that specify the optical anisotropy of the medium, and either the scattering properties of the inclusion or the lifetime of the small fluorophore in the case of the time-resolved fluorescence experimental configuration.     

Color schemes to represent the orientation of anisotropic tissues from diffusion tensor data: application to white matter fiber tract mapping in the human brain.

This paper investigates the use of color to represent the directional information contained in the diffusion tensor. Ideally, one wants to take into account both the properties of human color vision and of the given display hardware to produce a representation in which differences in the orientation of anisotropic structures are proportional to the perceived differences in color. It is argued here that such a goal cannot be achieved in general and therefore, empirical or heuristic schemes, which avoid some of the common artifacts of previously proposed approaches, are implemented. Directionally encoded color (DEC) maps of the human brain obtained using these schemes clearly show the main association, projection, and commissural white matter pathways. In the brainstem, motor and sensory pathways are easily identified and can be differentiated from the transverse pontine fibers and the cerebellar peduncles. DEC maps obtained from diffusion tensor imaging data provide a simple and effective way to visualize fiber direction, useful for investigating the structural anatomy of different organs. Magn Reson Med 42:526-540, 1999.     

Postprandial Hypotension and Impaired Postprandial Sustained and Selective Attention in Older Inpatients: Is There a Link?

ObjectivesThe study aimed to investigate the prevalence of postprandial hypotension (PPH) in older inpatients, to verify the overall postprandial behavior of blood pressure and attentional performances, and to explore the overall associations between blood pressure (including PPH) and attentional performances. Eventually, we aimed to investigate differences on PPH, blood pressure values and attentional performances based on the subjects’ frailty status.DesignCross-sectional study.Setting and ParticipantsA sample of older inpatients at the Geriatric Unit of the University Hospital of Messina (Italy).MethodsBasal, preprandial, and postprandial blood pressures (75 minutes after the meal) were measured for each patient; PPH was detected according to its empirical definition. Global cognitive functioning, and sustained and selective attention were assessed; a 46-item Frailty Index was calculated.ResultsThe sample consisted of 112 inpatients (54 females), with a mean age of 80.9 years. The prevalence of PPH was 30.4%; in the postprandial window, a reduction in blood pressure between 10 and 20 mm Hg and a reduction of >20 mm Hg were reported by 27.1% and 29.9% of inpatients, respectively. In the postprandial evaluation, sustained and selective attention markedly decreased. No significant associations were found between PPH occurrence and the postprandial dip of attentional performances, and no significant cognitive differences were found between inpatients with and without PPH. On the other hand, reduced postprandial attentional performances were associated especially with preprandial lower systolic and diastolic blood pressure values. Ultimately, no significant differences in PPH occurrence were found between frail and nonfrail inpatients; frail inpatients significantly exhibited also an overall lower cognitive functioning.Conclusions and ImplicationsIn our sample, PPH and impaired postprandial attentional performances were not associated, even though this association deserves further investigation. In hospitalized older adults, the accurate management of blood pressure levels appears relevant, because we evidenced that low blood pressure (especially preprandial) was associated with poor attentional functioning. Although the plausible occurrence of several interfering and confounder factors was observed in an acute care setting, we consider that the screening of attentional functioning among hospitalized older patients could be helpful.     

Evaluation of short-term and residual effects of Kinesio taping in chronic lateral epicondylitis: A randomized,double-blinded,controlled trial

BackgroundLateral epicondylitis is degenerative tendinosis of the extensor carpi radialis brevis muscle and is the most common work/sports-related chronic musculoskeletal problem affecting the elbow.PurposeThis study aimed to evaluate the short term and residual effectiveness of the Kinesio taping method on pain, grip force, quality of life, and functionality.Study DesignRandomized, double-blinded, controlled study.MethodsSubjects were 50 patients diagnosed with chronic unilateral lateral epicondylitis with a symptom duration of at least 12 weeks. During the first four weeks, the study group received a true inhibitor Kinesio taping while the control group received sham taping. In both groups, progressive stretching and strengthening exercises were given as a home program for six weeks. The primary outcome measure was the Numerical Rating Scale (NRS) for self-report of pain intensity; secondary outcome measures were Cyriax resistive muscle test evaluation, maximal grip strength, Patient- Rated Tennis Elbow Evaluation (PRTEE), and Short Form-36 (SF-36). After the treatment, patients were evaluated by the first assessor who was blinded to taping types.ResultsThere was a significant decrease in NRS scores overtime during the first four weeks in both groups (P < .001,) and effect sizes were large. There was no significant difference in Cyriax muscle resistance test maximal grip strength between groups (P > .05). However, there was a significant improvement in muscle strength of elbow extension and pronation in the study group detected in the intragroup analysis. Intragroup comparisons also showed a significant improvement in all subunits of the PRTEE and SF-36 except energy/vitality, social functioning, and pain in both groups (P < .05) with moderate to high effect sizes. PRTEE pain scores were significantly decreased in the study group compared to the placebo group (P < .05, d = 0.48).ConclusionThe effects of Kinesio taping on muscle strength, quality of life, and function in chronic lateral epicondylitis are not superior to placebo. However, NRS scores showed that in the two weeks after Kinesio taping treatment, pain reduction persisted as a residual effect which may improve the exercise adherence and functionality.     

A bacterial signature-based method for the identification of seven forensically relevant human body fluids

Detection and identification of body fluids plays a crucial role in criminal investigation, as it provides information on the source of the DNA as well as corroborative evidence regarding the crime committed, scene, and/or association with persons of interest. Historically, forensic serological methods have been chemical, immunological, catalytic, spectroscopic, and/or microscopic in nature. However, most of these methods are presumptive, with few robust confirmatory exceptions. In recent years several new molecular methods (mRNA, miRNA, DNA methylation, etc.) have been proposed; although promising, these methods require high quality human DNA or RNA. Additional steps are required in RNA based methods. Additionally, RNA based methods cannot be used for old cases where only DNA extracts remain to sample from. In this study, a novel non-human DNA (microbiome) based method was developed for the identification of the majority of forensically relevant human biological samples. Eight hundred and twelve (n = 812) biological samples (semen, vaginal fluid, menstrual blood, saliva, feces, urine, and blood) were collected and preserved using methods commonly used in forensic laboratories for evidence collection. Variable region four (V4) of 16 S ribosomal DNA (16 S rDNA) was amplified using a dual-indexing strategy and then sequenced on the MiSeq FGx sequencing platform using the MiSeq Reagent Kit v2 (500 cycles) and following the manufacturer’s protocol. Machine learning prediction models were used to assess the classification accuracy of the newly developed method. As there was no significant difference in bacterial communities between vaginal fluid, menstrual blood, and female urine, these were combined as female intimate samples. Except in urine, the bacterial structures associated with male and female body fluid samples were not significantly different from one another. The newly developed method accurately identified human body fluid samples with an overall accuracy of more than 88%. This newly developed bacterial signature-based method is fast (no additional steps are needed as the same DNA can be used for both body fluid identification and STR typing), efficient (consume less sample as a single test can identify all major body fluids), sensitive (needs only 5 pg of bacterial DNA), accurate, and can be easily added into a forensic high throughput sequencing (HTS) panel.     

TeleNEwCARe: An Italian case-control telegenetics study in patients with Hereditary NEuromuscular and CARdiac diseases

Telemedicine provides healthcare services remotely and represents a fundamental resource for the management of rare and fragile patients. Tele-health implementation is a main objective of the European Reference Networks (ERNs) mission to accelerate diagnosis for rare diseases.TeleNewCARe is a pilot case-control project which evaluates the efficacy and satisfaction of telegenetics for neuromuscular and cardiac adult patients, compared to face-to-face genetic counselling. The virtual sessions were co-hosted by a medical geneticist and a neurologist/cardiologist. Specific questionnaires (Clinical Genetics Satisfaction Questionnaire (CGS), Telemedicine Satisfaction Questionnaire (TSQ) and a Satisfaction Questionnaire for medical geneticists) were used to assess the effectiveness and fulfilment of telecounselling, both for patients and health care providers.Satisfaction expressed for telegenetics did not significantly differ from face-to-face counselling. The virtually enrolled patients declared they had the possibility to relate confidentially with the specialists, to share information and to be informed in an exhaustive way about their disease. Almost all patients declared themselves willing to reuse the telecounselling in the future. The multidisciplinary care was perceived as a significant added value. No overt technical problems were reported although the need for digital skills and tools can limit patients’ compliance.Our experience supports telegenetics as a valid alternative to traditional genetic counselling in cardiac and neuromuscular patients. This innovative approach facilitates multidisciplinary care, grants a periodical follow up, without forcing patients to discomfortable travelling, and allows to maintain expert care. This result meets the ERNs needs to reduce patients’ burden to access and monitor their healthcare.