Forensic species identification: practical guide for animal and plant DNA analysis.

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed.

Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles.

PEGylated lipid nanoparticles (LNPs) are commonly used to deliver bioactive molecules, but the role of PEGylation in DNA-loaded LNP interactions at the cellular and subcellular levels remains poorly understood. In this study, we investigated the mechanism of action of DNA-loaded PEGylated LNPs using gene reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), and fluorescence confocal microscopy (FCS). We found that PEG has no significant impact on the size or nanostructure of DNA LNPs but reduces their zeta potential and interaction with anionic cell membranes. PEGylation increases the structural stability of LNPs and results in lower DNA unloading. FCS experiments revealed that PEGylated LNPs are internalized intact inside cells and largely shuttled to lysosomes, while unPEGylated LNPs undergo massive destabilization on the plasma membrane. These findings can inform the design, optimization, and validation of DNA-loaded LNPs for gene delivery and vaccine development.

Two caseworks for one gene: successful species identification from compromised bone materials with the 12S rRNA.

The availability of a reliable molecular assay in species recognition in forensic cases is of paramount importance when visual inspection or morphological methods are not exhaustive, especially from challenging samples. Here, two different caseworks involving bone samples founded during medico-legal outdoor investigations are presented. In order to exclude the human nature of the specimens and to determine the exact species they belong to, we proceeded with the molecular approach trying to generate sequences from the classical mtDNA markers cyt b and COI. However, they both gave critical results. For this reason, a short amplicon of ~ 150 bp of the 12S rRNA gene was used as an alternative.This short fragment was sufficient to identify the biological origin of the bone specimens with a high degree of certainty leading to the exclusion of their human nature. This work highlights the utility of the 12S rRNA and underlines the importance of deepen the choice of alternative shorter markers with respect to the classical ones, in order to achieve species identification even from challenging and degraded material in forensic criminal and wildlife caseworks.

Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking.

Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.

Y-chromosome variability and genetic history of Commons from Northern Italy.

OBJECTIVES: Genetic drift and admixture are driving forces in human evolution, but their concerted impact to population evolution in historical times and at a micro-geographic scale is poorly assessed. In this study we test a demographic model encompassing both admixture and drift to the case of social-cultural isolates such as the so-called "Commons." MATERIALS AND METHODS: Commons are peculiar institutions of medieval origins whose key feature is the tight relationship between population and territory, mediated by the collective property of shared resources. Here, we analyze the Y-chromosomal genetic structure of four Commons (for a total of 366 samples) from the Central and Eastern Padana plain in Northern Italy. RESULTS: Our results reveal that all these groups exhibit patterns of significant diversity reduction, peripheral/outlier position within the Italian/European genetic space and high frequency of Common-specific haplogroups. By explicitly testing different drift-admixture models, we show that a drift-only model is more probable for Central Padana Commons, while additional admixture (~20%) from external population around the same time of their foundation cannot be excluded for the Eastern ones. DISCUSSION: Building on these results, we suggest central Middle Ages as the most probable age of foundation for three of the considered Commons, the remaining one pointing to late antiquity. We conclude that an admixture-drift model is particularly useful for interpreting the genetic structure and recent demographic history of small-scale populations in which social-cultural features play a significant role.

Insights into Punic genetic signatures in the southern necropolis of Tharros (Sardinia).

BACKGROUND: Phoenician and Punic expansions have been protagonists of intense trade networks and settlements in the Mediterranean Sea. AIMS: The maternal genetic variability of ancient Punic samples from the Sardinian necropolis of Tharros was analysed, with the aim to explore genetic interactions and signatures of past population events. SUBJECTS AND METHODS: The mtDNA HVS-I and coding region SNPs were analysed in 14 Punic samples and 74 modern individuals from Cabras and Belvì (for which the HVS-II region was also analysed). The results were compared with 5,590 modern Euro-Mediterranean sequences and 127 ancient samples. RESULTS: While contemporary groups fall within the genetic variability of other modern Sardinians, our Punic samples reveal proximity to present-day North-African and Iberian populations. Furthermore, Cabras and Belvì cluster mainly with pre-Phoenician groups, while samples from Tharros project with other Punic Sardinian individuals. CONCLUSION: This study provides the first preliminary insights into the population dynamics of the Punic site of Tharros. While the number of currently available samples does not allow definitive investigation of the connection with indigenous Sardinian groups, our results seem to confirm internal migratory phenomena in the central-western Mediterranean and female participation in the Punic mobility.

ß-Cell Pathophysiology: A Review of Advanced Optical Microscopy Applications.

ß-cells convert glucose (input) resulting in the controlled release of insulin (output), which in turn has the role to maintain glucose homeostasis. ß-cell function is regulated by a complex interplay between the metabolic processing of the input, its transformation into second-messenger signals, and final mobilization of insulin-containing granules towards secretion of the output. Failure at any level in this process marks ß-cell dysfunction in diabetes, thus making ß-cells obvious potential targets for therapeutic purposes. Addressing quantitatively ß-cell (dys)function at the molecular level in living samples requires probing simultaneously the spatial and temporal dimensions at the proper resolution. To this aim, an increasing amount of research efforts are exploiting the potentiality of biophysical techniques. In particular, using excitation light in the visible/infrared range, a number of optical-microscopy-based approaches have been tailored to the study of ß-cell-(dys)function at the molecular level, either in label-free mode (i.e., exploiting intrinsic autofluorescence of cells) or by the use of organic/genetically-encoded fluorescent probes. Here, relevant examples from the literature are reviewed and discussed. Based on this, new potential lines of development in the field are drawn.

The genetic legacy of the Yaghnobis: A witness of an ancient Eurasian ancestry in the historically reshuffled central Asian gene pool.

OBJECTIVES: The Yaghnobis are an ethno-linguistic minority historically settled along the Yaghnob River in the Upper-Zarafshan Valley in Tajikistan. They speak a language of Old Sogdian origin, which is the only present-day witness of the Lingua Franca used along the Silk Road in Late Antiquity. The aim of this study was to reconstruct the genetic history of this community in order to shed light on its isolation and genetic ancestry within the Euro-Asiatic context. MATERIALS AND METHODS: A total of 100 DNA samples were collected in the Yaghnob and Matcha Valleys during several expeditions and their mitochondrial, Y-chromosome and autosomal genome-wide variation were compared with that from a large set of modern and ancient Euro-Asiatic samples. RESULTS: Findings from uniparental markers highlighted the long-term isolation of the Yaghnobis. Mitochondrial DNA ancestry traced an ancient link with Middle Eastern populations, whereas Y-chromosome legacy showed more tight relationships with Central Asians. Admixture, outgroup-f3, and D-statistics computed on autosomal variation corroborated Y-chromosome evidence, pointing respectively to low Anatolian Neolithic and high Steppe ancestry proportions in Yaghnobis, and to their closer affinity with Tajiks than to Iranians. DISCUSSION: Although the Yaghnobis do not show evident signs of recent admixture, they could be considered a modern proxy for the source of gene flow for many Central Asian and Middle Eastern groups. Accordingly, they seem to retain a peculiar genomic ancestry probably ascribable to an ancient gene pool originally wide spread across a vast area and subsequently reshuffled by distinct demographic events occurred in Middle East and Central Asia.

Probing the light scattering properties of insulin secretory granules in single live cells.

Light scattering was recently demonstrated to serve as an intrinsic indicator for pancreatic islet cell mass and secretion. The insulin secretory granule (ISG), in particular, was proposed to be a reasonable candidate as the main intracellular source of scattered light due to the densely-packed insulin semi-crystal in the granule lumen. This scenario, if confirmed, would in principle open new perspectives for label-free single-granule imaging, tracking, and analysis. Contrary to such expectations, here we demonstrate that ISGs are not a primary source of scattering in primary human ß-cells, as well as in immortalized ß-like cells, quantitatively not superior to other intracellular organelles/structures, such as lysosomes and internal membranes. This result is achieved through multi-channel imaging of scattered light along with fluorescence arising from selectively-labelled ISGs. Co-localization and spatiotemporal cross-correlation analysis is performed on these signals, and compared among different cell lines. Obtained results suggest a careful re-thinking of the possibility to exploit intrinsic optical properties originating from ISGs for single-granule imaging purposes.

Iron Age Italic population genetics: the Piceni from Novilara (8th-7th century BC).

BACKGROUND: Archaeological data provide evidence that Italy, during the Iron Age, witnessed the appearance of the first communities with well defined cultural identities. To date, only a few studies report genetic data about these populations and, in particular, the Piceni have never been analysed. AIMS: To provide new data about mitochondrial DNA (mtDNA) variability of an Iron Age Italic population, to understand the contribution of the Piceni in shaping the modern Italian gene pool and to ascertain the kinship between some individuals buried in the same grave within the Novilara necropolis. SUBJECTS AND METHODS: In a first set of 10 individuals from Novilara, we performed deep sequencing of the HVS-I region of the mtDNA, combined with the genotyping of 22 SNPs in the coding region and the analysis of several autosomal markers. RESULTS: The results show a low nucleotide diversity for the inhabitants of Novilara and highlight a genetic affinity of this ancient population with the current inhabitants of central Italy. No family relationship was observed between the individuals analysed here. CONCLUSIONS: This study provides a preliminary characterisation of the mtDNA variability of the Piceni of Novilara, as well as a kinship assessment of two peculiar burials.

Static and moving frontiers: the genetic landscape of Southern African Bantu-speaking populations.

A consensus on Bantu-speaking populations being genetically similar has emerged in the last few years, but the demographic scenarios associated with their dispersal are still a matter of debate. The frontier model proposed by archeologists postulates different degrees of interaction among incoming agropastoralist and resident foraging groups in the presence of "static" and "moving" frontiers. By combining mitochondrial DNA and Y chromosome data collected from several southern African populations, we show that Bantu-speaking populations from regions characterized by a moving frontier developing after a long-term static frontier have larger hunter-gatherer contributions than groups from areas where a static frontier was not followed by further spatial expansion. Differences in the female and male components suggest that the process of assimilation of the long-term resident groups into agropastoralist societies was gender biased. Our results show that the diffusion of Bantu languages and culture in Southern Africa was a process more complex than previously described and suggest that the admixture dynamics between farmers and foragers played an important role in shaping the current patterns of genetic diversity.

Between Andes and Amazon: the genetic profile of the Arawak-speaking Yanesha.

The Yanesha are a Peruvian population who inhabit an environment transitional between the Andes and Amazonia. They present cultural traits characteristic of both regions, including in the language they speak: Yanesha belongs to the Arawak language family (which very likely originated in the Amazon/Orinoco lowlands), but has been strongly influenced by Quechua, the most widespread language family of the Andes. Given their location and cultural make-up, the Yanesha make for an ideal case study for investigating language and population dynamics across the Andes-Amazonia divide. In this study, we analyze data from high and mid-altitude Yanesha villages, both Y chromosome (17 STRs and 16 SNPs diagnostic for assigning haplogroups) and mtDNA data (control region sequences and 3 SNPs and one INDEL diagnostic for assigning haplogroups). We uncover sex-biased genetic trends that probably arose in different stages: first, a male-biased gene flow from Andean regions, genetically consistent with highland Quechua-speakers and probably dating back to Inca expansion; and second, traces of European contact consistent with Y chromosome lineages from Italy and Tyrol, in line with historically documented migrations. Most research in the history, archaeology and linguistics of South America has long been characterized by perceptions of a sharp divide between the Andes and Amazonia; our results serve as a clear case-study confirming demographic flows across that 'divide'.

Increased efficiency in geographic ancestry assignment and human identification by combining lineage profiles: The case of the Iranians.

OBJECTIVES: This research is a first empirical attempt to quantify the increase of the among-groups variance and the probative value of a DNA evidence when combining profiles based on markers with uniparental inheritance. METHODS: Yfiler and HVS-I panels of loci were analyzed in 130 healthy unrelated males from six Iranian native groups. RESULTS: A separate analysis of DNA profiles at the two lineage markers failed to detect a population substructure, whereas maximum levels of genetic diversity (HD = 1) and discrimination capacity (DC = 1) were obtained by combining the two profiles. CONCLUSIONS: When combined, the forensic efficiency of routinely used panels of lineage markers can be largely sufficient to resolve cases of geographic ancestry and human identification even in genetically homogeneous populations.

Spatiotemporal oxygen dynamics in young leaves reveal cyclic hypoxia in plants.

Oxygen is essential for plant growth and development. Hypoxia occurs in plants due to limited oxygen availability following adverse environmental conditions as well in hypoxic niches in otherwise normoxic environments. However, the existence and functional integration of spatiotemporal oxygen dynamics with plant development remains unknown. In animal systems dynamic fluctuations in oxygen availability are known as cyclic hypoxia. In this study, we demonstrate that cyclic fluctuations in internal oxygen levels occur in young emerging leaves of Arabidopsis plants. Cyclic hypoxia in plants is based on a mechanism requiring the ETHYLENE RESPONSE FACTORS type VII (ERFVII) that are central components of the oxygen-sensing machinery in plants. The ERFVII-dependent mechanism allows precise adjustment of leaf growth in response to carbon status and oxygen availability within plant cells. This study thus establishes a functional connection between internal spatiotemporal oxygen dynamics and developmental processes of plants.

Exploring Regorafenib Responsiveness and Uncovering Molecular Mechanisms in Recurrent Glioblastoma Tumors through Longitudinal In Vitro Sampling.

Glioblastoma, a deadly brain tumor, shows limited response to standard therapies like temozolomide (TMZ). Recent findings from the REGOMA trial underscore a significant survival improvement offered by Regorafenib (REGO) in recurrent glioblastoma. Our study aimed to propose a 3D ex vivo drug response precision medicine approach to investigate recurrent glioblastoma sensitivity to REGO and elucidate the underlying molecular mechanisms involved in tumor resistance or responsiveness to treatment. Three-dimensional glioblastoma organoids (GB-EXPs) obtained from 18 patients' resected recurrent glioblastoma tumors were treated with TMZ and REGO. Drug responses were evaluated using NAD(P)H FLIM, stratifying tumors as responders (Resp) or non-responders (NRs). Whole-exome sequencing was performed on 16 tissue samples, and whole-transcriptome analysis on 13 GB-EXPs treated and untreated. We found 35% (n = 9) and 77% (n = 20) of tumors responded to TMZ and REGO, respectively, with no instances of TMZ-Resp being REGO-NRs. Exome analysis revealed a unique mutational profile in REGO-Resp tumors compared to NR tumors. Transcriptome analysis identified distinct expression patterns in Resp and NR tumors, impacting Rho GTPase and NOTCH signaling, known to be involved in drug response. In conclusion, recurrent glioblastoma tumors were more responsive to REGO compared to TMZ treatment. Importantly, our approach enables a comprehensive longitudinal exploration of the molecular changes induced by treatment, unveiling promising biomarkers indicative of drug response.

Signatures of the preagricultural peopling processes in sub-Saharan Africa as revealed by the phylogeography of early Y chromosome lineages.

The study of Y chromosome variation has helped reconstruct demographic events associated with the spread of languages, agriculture, and pastoralism in sub-Saharan Africa, but little attention has been given to the early history of the continent. In order to overcome this lack of knowledge, we carried out a phylogeographic analysis of haplogroups A and B in a broad data set of sub-Saharan populations. These two lineages are particularly suitable for this objective because they are the two most deeply rooted branches of the Y chromosome genealogy. Their distribution is almost exclusively restricted to sub-Saharan Africa where their frequency peaks at 65% in groups of foragers. The combined high-resolution single nucleotide polymorphism analysis with short tandem repeats variation of their subclades reveals strong geographic and population structure for both haplogroups. This has allowed us to identify specific lineages related to regional preagricultural dynamics in different areas of sub-Saharan Africa. In addition, we observed signatures of relatively recent contact, both among Pygmies and between them and Khoisan speaker groups from southern Africa, thus contributing to the understanding of the complex evolutionary relationships among African hunter-gatherers. Finally, by revising the phylogeography of the very early human Y chromosome lineages, we have obtained support for the role of southern Africa as a sink, rather than a source, of the first migrations of modern humans from eastern and central parts of the continent. These results open new perspectives on the early history of Homo sapiens in Africa, with particular attention to areas of the continent where human fossil remains and archaeological data are scant.

The peopling of Europe and the cautionary tale of Y chromosome lineage R-M269.

Recently, the debate on the origins of the major European Y chromosome haplogroup R1b1b2-M269 has reignited, and opinion has moved away from Palaeolithic origins to the notion of a younger Neolithic spread of these chromosomes from the Near East. Here, we address this debate by investigating frequency patterns and diversity in the largest collection of R1b1b2-M269 chromosomes yet assembled. Our analysis reveals no geographical trends in diversity, in contradiction to expectation under the Neolithic hypothesis, and suggests an alternative explanation for the apparent cline in diversity recently described. We further investigate the young, STR-based time to the most recent common ancestor estimates proposed so far for R-M269-related lineages and find evidence for an appreciable effect of microsatellite choice on age estimates. As a consequence, the existing data and tools are insufficient to make credible estimates for the age of this haplogroup, and conclusions about the timing of its origin and dispersal should be viewed with a large degree of caution.

The dual origin of Tati-speakers from Dagestan as written in the genealogy of uniparental variants.

OBJECTIVES: Tat language is classified in an Iranian subbranch of the Indo-European family. It is spoken in the Caucasus and in the West Caspian region by populations with heterogeneous cultural traditions and religion whose ancestry is unknown. The aim of this study is to get a first insight about the genetic history of this peculiar linguistic group. METHODS: We investigated the uniparental gene pools, defined by NRY and mtDNA high-resolution markers, in two Tati-speaking communities from Dagestan: Mountain Jews or Juhur, who speak the Judeo-Tat dialect, and the Tats, who speak the Muslim-Tat dialect. The samples have been collected in monoethnic rural villages and selected on the basis of genealogical relationships. A novel approach aimed at resolving cryptic cases in the recent history of human populations, which combines the properties of uniparental genetic markers with the potential of "forward-in-time" computer simulations, is presented. RESULTS: Judeo-Tats emerged as a group with tight matrilineal genetic legacy who separated early from other Jewish communities. Tats exhibited genetic signals of a much longer in situ evolution, which appear as substantially unlinked with other Indo-Iranian enclaves in the Caucasus. CONCLUSIONS: The independent demographic histories of the two samples, with mutually reversed profiles at paternally and maternally transmitted genetic systems, suggest that geographic proximity and linguistic assimilation of Tati-speakers from Dagestan do not reflect a common ancestry.

Single-cell imaging of α and ß cell metabolic response to glucose in living human Langerhans islets.

Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and ß cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 ß cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and ß cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and ß cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and ß cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power.

In vitro and ex vivo nano-enabled immunomodulation by the protein corona.

New technologies with the capacity to tune immune system activity are highly desired in clinical practice and disease management. Here we demonstrate that nanoparticles with a protein corona enriched with gelsolin (GSN), an abundant plasma protein that acts as a modulator of immune responses, are avidly captured by human monocytic THP-1 cells in vitro and by leukocyte subpopulations derived from healthy donors ex vivo. In human monocytes, GSN modulates the production of tumor necrosis factor alpha (TNF-α) in an inverse dose-dependent manner. Overall, our results suggest that artificial coronas can be exploited to finely tune the immune response, opening new approaches for the prevention and treatment of diseases.