Taking the microfluidic approach to nucleic acid analysis in forensics: Review and perspectives.

Forensic laboratories are universally acknowledged as being overburdened, underfunded, and in need of improved analytical methods to expedite investigations, decrease the costs associated with nucleic acid (NA) analysis, and perform human identification (HID) at the point of need (e.g., crime scene, booking station, etc.). In response, numerous research and development (R&D) efforts have resulted in microfluidic tools that automate portions of the forensic genetic workflow, including DNA extraction, amplification, and short tandem repeat (STR) typing. By the early 2000 s, reports from the National Institute of Justice (NIJ) anticipated that microfluidic 'swab-in-profile-out' systems would be available for use at the crime scene by 2015 and the FBI's 2010 'Rapid DNA' Initiative, approved by Congress in 2017, directed this effort by guiding the development and implementation of maturing systems. At present, few fully-automated microfluidic DNA technologies are commercially available for forensic HID and their adoption by agencies interested in identification has been limited. In practice, the integration of complex laboratory processes to produce one autonomous unit, along with the highly variable nature of forensic input samples, resulted in systems that are more expensive per sample and not comparable to gold-standard identification methods in terms of sensitivity, reproducibility, and multiplex capability. This Review and Perspective provides insight into the contributing factors to this outcome; namely, we focus on the complications associated with the tremendous undertaking that is developing a sample-in-answer-out platform for HID. For context, we also describe the intricate forensic landscape that contributes to a nuanced marketplace, not easily distilled down to cases of simple supply and demand. Moving forward and considering the trade-offs associated with developing methods to compete, sometimes directly, with conventional ones, we recommend a focus shift for microfluidics developers toward the creation of innovative solutions for emerging applications in the field to increase the bandwidth of the forensic investigative toolkit. Likewise, we urge case working personnel to reframe how they conceptualize the currently available Rapid DNA tools; rather than comparing these microfluidic methods to gold-standard procedures, take advantage of their rapid and integrated modes for those situations requiring expedited identifications in an informed manner.

Rapid Microchip Electrophoretic Separation of Novel Transcriptomic Body Fluid Markers for Forensic Fluid Profiling.

Initial screening of criminal evidence often involves serological testing of stains of unknown composition and/or origin discovered at a crime scene to determine the tissue of origin. This testing is presumptive but critical for contextualizing the scene. Here, we describe a microfluidic approach for body fluid profiling via fluorescent electrophoretic separation of a published mRNA panel that provides unparalleled specificity and sensitivity. This centrifugal microfluidic approach expedites and automates the electrophoresis process by allowing for simple, rotationally driven flow and polymer loading through a 5 cm separation channel; with each disc containing three identical domains, multi-sample analysis is possible with a single disc and multi-sample detection per disc. The centrifugal platform enables a series of sequential unit operations (metering, mixing, aliquoting, heating, storage) to execute automated electrophoretic separation. Results show on-disc fluorescent detection and sizing of amplicons to perform comparably with a commercial 'gold standard' benchtop instrument and permitted sensitive, empirical discrimination between five distinct body fluids in less than 10 min. Notably, our microfluidic platform represents a faster, simpler method for separation of a transcriptomic panel to be used for forensically relevant body fluid identification.

Ultra-rapid real-time microfluidic RT-PCR instrument for nucleic acid analysis.

The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the 'gold standard'. While instrumentation for executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.

Sensitivity of serology assay in Covid-19 diagnosis: does the antigen matter?

Since the spreading of Sar-CoV-2 in March 2020, many serologic tests have been developed to identify antibody responses. Indeed, different commercial kits are directed against different antigens and could utilise different methods thereby triggering confusion and criticism. Here, we compared two Food and Drug Administration (FDA)-approved automatized assays that detect IgG responses against spike or nucleocapsid protein of Sars-Cov-2 virus in 127 subjects among healthcare workers of IRCCS Policlinico San Donato (MI), Italy. We observed different kinetics of IgG responses, demonstrating the importance of timing of sampling to correctly interpret the results both for infection diagnosis and for epidemiologic studies. We observed that Anti-N response starts earlier than Anti-S1/S2 response but also decreases earlier, affecting the sensitivity of the tests at different time points. Combining two different assays, designed against different antigens, could reduce false negative results. Finally, we observed a patient who produced anti-nucleocapsid IgG, but not anti-spike IgG. In conclusion, we investigated antibody responses in Covid-19 disease, aiming to direct clinicians and laboratory scientists to correctly interpret serologic results by always paying attention to clinical history correlation, timing of sampling, methods and antigens used, to avoid false negative results and obtain relevant epidemiologic data.

The mysterious life of the plant trans-Golgi network: advances and tools to understand it better.

By being at the interface of the exocytic and endocytic pathways, the plant trans-Golgi network (TGN) is a multitasking and highly diversified organelle. Despite governing vital cellular processes, the TGN remains one of the most uncharacterized organelle of plant cells. In this review, we highlight recent studies that have contributed new insights and to the generation of markers needed to answer several important questions on the plant TGN. Several drugs specifically affecting proteins critical for the TGN functions have been extremely useful for the identification of mutants of the TGN in the pursuit to understand how the morphology and the function of this organelle are controlled. In addition to these chemical tools, we review emerging microscopy techniques that help visualize the TGN at an unpreceded resolution and appreciate the heterogeneity and dynamics of this organelle in plant cells.

Premature senescence in primary muscle cultures of myotonic dystrophy type 2 is not associated with p16 induction.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic disorders linked to two different genetic loci and characterized by several features including myotonia, muscle weakness and atrophy, cardiac dysfunctions, cataracts and insulin-resistance. In both forms, expanded nucleotide sequences cause the accumulation of mutant transcripts in the nucleus deregulating the activity of some RNAbinding proteins and providing an explanation for the multisystemic phenotype of DM patients. However this pathogenetic mechanism does not explain some histopathological features of DM skeletal muscle like muscle atrophy. It has been observed that DM muscle shares similarities with the ageing muscle, where the progressive muscle weakness and atrophy is accompanied by a lower regenerative capacity possibly due to the failure in satellite cells activation. The aim of our study is to investigate if DM2 satellite cell derived myoblasts exhibit a premature senescence as reported for DM1 and if alterations in their proliferation potential and differentiation capabilities might contribute to some of the histopathological features observed in DM2 muscles. Our results indicate that DM myoblasts have lower proliferative capability than control myoblasts and reach in vitro senescence earlier than controls. Differentely from DM1, the p16 pathway is not responsible for the premature growth arrest observed in DM2 myoblasts which stop dividing with telomeres shorter than controls. During in vitro senescence, a progressive decrease in fusion index is observable in both DM and control myotubes with no significant differences between groups. Moreover, myotubes obtained from senescent myoblasts appear to be smaller than those from young myoblasts. Taken together, our data indicate a possible role of DM2 premature myoblast senescence in skeletal muscle histopathological alterations i.e., dystrophic changes and type 2 fibre atrophy.

Cultured myoblasts from patients affected by myotonic dystrophy type 2 exhibit senescence-related features: ultrastructural evidence.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant disorder caused by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc Finger Protein-9 gene. In DM2 tissues, the expanded mutant transcripts accumulate in nuclear focal aggregates where splicing factors are sequestered, thus affecting mRNA processing. Interestingly, the ultrastructural alterations in the splicing machinery observed in the myonuclei of DM2 skeletal muscles are reminiscent of the nuclear changes occurring in age-related muscle atrophy. Here, we investigated in vitro structural and functional features of satellite cell-derived myoblasts from biceps brachii, in the attempt to investigate cell senescence indices in DM2 patients by ultrastructural cytochemistry. We observed that in satellite cell-derived DM2 myoblasts, cell-senescence alterations such as cytoplasmic vacuolization, reduction of the proteosynthetic apparatus, accumulation of heterochromatin and impairment of the pre-mRNA maturation pathways occur earlier than in myoblasts from healthy patients. These results, together with preliminary in vitro observations on the early onset of defective structural features in DM2 myoblast derived-myotubes, suggest that the regeneration capability of DM2 satellite cells may be impaired, thus contributing to the muscular dystrophy in DM2 patients.

Spatiotemporal dynamics of the electrical network activity in the root apex.

The study of electrical network systems, integrated with chemical signaling networks, is becoming a common trend in contemporary biology. Classical techniques are limited to the assessment of signals from doublets or triplets of cells at a fixed temporal bin width. At present, full characteristics of the electrical network distribution and dynamics in plant cells and tissues has not been established. Here, a 60-channels multielectrode array (MEA) is applied to study spatiotemporal characteristics of the electrical network activity of the root apex. Both intense spontaneous electrical activities and stimulation-elicited bursts of locally propagating electrical signals have been observed. Propagation of the spikes indicates the existence of excitable traveling waves in plants, similar to those observed in non-nerve electrogenic tissues of animals. Obtained data reveal synchronous electric activities of root cells emerging in a specific root apex region. The dynamic electrochemical activity of root apex cells is proposed to continuously integrate internal and external signaling for developmental adaptations in a changing environment.

Asymmetrical dripping.

Dripping from a faucet is studied, where by cutting diagonally the tip breaks the cylindrical symmetry. We measured long dripping time series recorded with a laser-beam apparatus and continuous change in drop position using a high-speed camera. It is found that the added degree of freedom produces a transversal oscillation of a pending drop, which couples with a vertical oscillation induced by the breaking off of the previous drop. As a consequence dripping times shorten and dripping patterns regularize. The effect is attributed to the "reduced" contact circle and to decreased frequency of the vertical oscillations of the residue. A very complex flow circulation pattern within the forming drop is observed. It is suggested that geometrical shape vibrations of the pending drop take place by the development of eddies of different amount, more effective in the vicinity of the highest edge of the tip, where they dissipate more slowly. This asymmetrical liquid flow is brought about by the slanting form of the orifice and overlaps with the axial oscillations.

Experimental study of dripping dynamics.

The dependence of dripping dynamics from physical properties of the nozzles is investigated. The analysis is performed by means of two complementary methods: (i) long dripping time series recorded with a standard laser-beam apparatus; and (ii) drop formations observed with a fast digital camcorder. Dripping from nozzles of different sizes is analyzed, and the formation of satellite drops is related to the preeminent physical parameter of control (flow rate). Quasielastic collisions between parent and satellite drops are observed.

Mass on a spring map for the dripping faucet at low flow rates.

An improved discrete map for the mass-on-a-spring model for the dripping faucet is used to reproduce the evolution of experimental dripping spectra at low flow rates. If an inverse dependence of drop mass on flow rate is supposed, a repeated evolution from period 1 to chaos is obtained. A comparison between discrete relaxation oscillator attractors and dissipative type-web map attractors is carried out. It is found that a dissipative web map accounts for some characteristics of the relaxation oscillator map, thus giving further tools for investigation.

Programmed cell death in colorectal carcinogenesis.

The most studied mechanism of malignant transformation has been cell proliferation. The relationship between programmed cell death (apoptosis), cell proliferation, and apoptosis regulatory genes (p53 and bcl-2), was studied in normal colonic epithelium, 26 sporadic adenomas both early and late, 25 FAP adenomas, and 34 carcinomas. We showed a decrease in programmed cell death and an increase in cell proliferation during the transition from adenoma to carcinoma. The increase of expression of p53 from early (10%) to late adenomas (87%) contrasted with the decrease of bcl-2 staining. Sixty-two per cent and 23% of carcinomas were reactive for p53 and bcl-2 respectively. Abnormal early activation of the bcl-2 gene, rather than late p53 gene mutation appears to be responsible for inhibition of apoptosis in colorectal carcinogenesis. bcl-2 was higher in FAP adenomas than in sporadic cases, and in carcinomas favouring the accumulation of long-living cells, which are more subject to mutation and thus cancerization.