Morphometric approaches to Cannabis evolution and differentiation from archaeological sites: interpreting the archaeobotanical evidence from bronze age Haimenkou, Yunnan.

Cannabis grains are frequently reported from archaeological sites in Asia, and hypothesized centers of origins are China and Central Asia. Chinese early cannabis remains are often interpreted as evidence of hemp fabric production, in line with early textual evidence describing ritualistic hemp cloth use and hemp cultivation as a grain crop. Modern measurements on cannabis varieties show distinct sizes between fibre or oil/fibre and psychoactive varieties, the former having larger seeds on average than the latter. This paper reviews the current macro-botanical evidence for cannabis across East, Central and South Asia and builds a comparative framework based on modern cannabis seed measurements to help identify cannabis use in the past, through the metric analysis of archaeologically preserved seeds. Over 800 grains of cannabis were retrieved from the 2008 excavation of Haimenkou, Yunnan, Southwest China, dating to between 1650 and 400 bc. These are compared with other known archaeological cannabis and interpreted through the metric framework. This offers a basis for exploration of the seed morphometrics potential to infer cannabis cultivation and diversification in uses. At Haimenkou, cannabis seeds size mostly plot in the range of overlapping psychoactive/fibre types; we therefore suggest that the cannabis assemblage from Haimenkou is indicative of a crop beginning to undergo evolution from its early domesticated form towards a diversified crop specialized for alternative uses, including larger oilseed/fibre adapted varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s00334-023-00966-6.

Archaeological and molecular evidence for ancient chickens in Central Asia.

The origins and dispersal of the chicken across the ancient world remains one of the most enigmatic questions regarding Eurasian domesticated animals. The lack of agreement concerning timing and centers of origin is due to issues with morphological identifications, a lack of direct dating, and poor preservation of thin, brittle bird bones. Here we show that chickens were widely raised across southern Central Asia from the fourth century BC through medieval periods, likely dispersing along the ancient Silk Road. We present archaeological and molecular evidence for the raising of chickens for egg production, based on material from 12 different archaeological sites spanning a millennium and a half. These eggshells were recovered in high abundance at all of these sites, suggesting that chickens may have been an important part of the overall diet and that chickens may have lost seasonal egg-laying.

Agriculture along the upper part of the Middle Zarafshan River during the first millennium AD: A multi-site archaeobotanical analysis.

The Zarafshan River runs from the mountains of Tajikistan and terminates in the sands of the Kyzyl-Kum Desert in Uzbekistan; it served as a communication route and homeland for the Sogdians. The Sogdians are historically depicted as merchants existing from the end of the first millennium BC through the first millennium AD. While recent research has provided the first glimpse into cultivation, commerce, communication, and consumption in the Lower Zarafshan, the agricultural heartland of the Middle Zarafshan Basin has remained unstudied. This paper presents the results of archaeobotanical investigations conducted at five ancient urban sites/areas spanning the fifth to the twelfth centuries AD: Kainar (Penjikent citadel), Penjikent (shahristan), Sanjar-Shah, Kuk-Tosh (pre-Mongol Penjikent), and Afrasiab. Collectively, these data show that cereals, legumes, oil/fiber crops, fruits, and nuts were cultivated on the fertile Zarafshan floodplains. In this paper, we discuss evidence for the diversification of the agricultural assemblage over time, including the introduction of new staple crops and fruits into an already complex cultivation system. In addition, we contrast our data with previously published results from sites along the course of the Zarafshan to determine whether there is a dietary difference between pre-and post-Islamic conquest periods at settlements located along the river.

Food production and agricultural systems on the southwestern frontier of the Han Empire: archaeobotanical remains from the 2016 excavation of Hebosuo, Yunnan.

Dian Basin in Yunnan province is an important center for both early agricultural production and centralized state formation. Settled agricultural villages are present in the province since at least the third millennium BC, and by the first millennium BC, the Dian Culture, a highly specialized bronze polity, flourished in the Dian Basin and surrounding area, until it was conquered by the Han in 109 BC. The increased deployment of flotation at recent archaeological excavations in Yunnan allowed the reconstruction of agricultural practices from the Neolithic to the early Bronze Age, documented at Baiyangcun, Haimenkou, and Xueshan among others. However, archaeobotanical evidence relating to the pivotal period right before and after the Han conquest have so far been lacking, with only limited written records about agricultural production in the Shiji by Sima Qian. Here we present for the first time direct archaeobotanical evidence relating to this transitional period as revealed by rich Han period deposits found during the 2016 excavation of Hebosuo, the largest Dian settlement investigated in Yunnan so far, dated by direct AMS on charred cereal grains and artefactual evidence as spanning from between 850 BC-220 AD. Following the Han conquest, the main components of the agricultural system did not undergo radical changes, but the weedy flora indicates a heavier reliance of wet-land rice systems, evidencing a higher level of water management or even irrigation practices, and the consequent intensification of the agricultural production. These findings on shifting agricultural regimes in Yunnan also contribute to current debates about the interplay between intensification, food risk, and ecology in times of political instability. Supplementary Information: The online version contains supplementary material available at 10.1007/s12520-023-01766-9.

The origins of multi-cropping agriculture in Southwestern China: Archaeobotanical insights from third to first millennium B.C. Yunnan.

Yunnan's location at the crossroad of temperate China, Northeast India and tropical mainland Southeast Asia makes it a pivotal area for the understanding of early cultural contacts and agricultural spread between these ecologically diverse regions. This paper evaluates current evidence relating to the emergence of the first agricultural systems in Yunnan. It also reviews previous theories on agricultural dispersal to Yunnan, including whether Austroasiatic speakers were responsible for the spread of rice from Yunnan to mainland Southeast Asia, and builds a new framework that allows to tie agricultural development in the region into broader patterns of early migration and exchange networks. Archaeobotanical remains attest to an initial spread of rice and millet from Central China into Yunnan in the third millennium B.C. and the establishment of a mixed-crop economy; the introduction of wheat and barley in the second millennium B.C. allowed for increased diversification of the agricultural system, with a two-season intensification trend in the late first millennium B.C. Differences in early rice cultivation ecologies between Yunnan and mainland Southeast Asia suggest that Yunnan rice farmers may not have had a primary role in the southern dispersal of rice, however, more data is needed to fully clarify the source and development of dryland cultivation of rice in mainland Southeast Asia. Supplementary Information: The online version contains supplementary material available at 10.1007/s41826-022-00052-2.

Quantitation of Poly(ADP-Ribose) by Isotope Dilution Mass Spectrometry.

Poly(ADP-ribosyl)ation (PARylation), i.e., the formation of the nucleic acid-like biopolymer poly(ADP-ribose) (PAR), is an essential posttranslational modification carried out by poly(ADP-ribose) polymerases (PARPs). While PAR levels are low under physiological conditions, they can transiently increase more than 100-fold upon induction of genotoxic stress. The accurate quantitation of cellular PAR with high sensitivity is of critical importance to understand the role of PARylation in cellular physiology and pathophysiology and to determine the pharmacodynamic efficiencies of clinically relevant PARP inhibitors, which represent a novel class of promising chemotherapeutics. Previously, we have developed a bioanalytical platform based on isotope dilution mass spectrometry (LC-MS/MS) to quantify cellular PAR with unequivocal chemical specificity in absolute terms with femtomol sensitivity (Martello et al. ACS Chem Biol 8(7):1567-1575, 2013). This method enables the analysis of steady-state levels, as well as stress-induced levels of PAR in various biological systems including cell lines, mouse tissues, and primary human lymphocytes. It has a wide range of potential applications in basic research, as well as in drug development (Martello et al. ACS Chem Biol 8(7):1567-1575, 2013; Mangerich et al. Toxicol Lett 244:56-71, 2016). Here, we present an improved and adjusted version of the original protocol by Martello/Mangerich et al., which uses UPLC-MS/MS instrumentation.

Proteome-Wide Identification of In Vivo ADP-Ribose Acceptor Sites by Liquid Chromatography-Tandem Mass Spectrometry.

ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography-high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.

Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue.

Although protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites. Here, we present an experimental workflow for sensitive and unbiased analysis of endogenous ADP-ribosylation sites, capable of detecting more than 900 modification sites in mammalian cells and mouse liver. In cells, we demonstrate that Lys residues, besides Glu, Asp and Arg residues, are the dominant in vivo targets of ADP-ribosylation during oxidative stress. In normal liver tissue, we find Arg residues to be the predominant modification site. The cellular distribution and biological processes that involve ADP-ribosylated proteins are different in cultured cells and liver tissue, in the latter of which the majority of sites were found to be in cytosolic and mitochondrial protein networks primarily associated with metabolism. Collectively, we describe a robust methodology for the assessment of the role of ADP-ribosylation and ADP-ribosyltransferases in physiological and pathological states.

Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of N7-ETE-guanine DNA adducts, the excision rate of CEES-induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD(+) levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES-induced short-term cytotoxicity 24h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi-functional mustards such as SM and HN2. In conclusion, we demonstrate that PARylation plays a functional role in mustard-induced cellular stress response with substance-specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM-related pathologies and to sensitize cancer cells for mustard-based chemotherapy, potential long-term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy.

Quantification of cellular poly(ADP-ribosyl)ation by stable isotope dilution mass spectrometry reveals tissue- and drug-dependent stress response dynamics.

Poly(ADP-ribosyl)ation is an essential post-translational modification with the biopolymer poly(ADP-ribose) (PAR). The reaction is catalyzed by poly(ADP-ribose) polymerases (PARPs) and plays key roles in cellular physiology and stress response. PARP inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality. We have developed an accurate and sensitive bioanalytical platform based on isotope dilution mass spectrometry in order to quantify steady-state and stress-induced PAR levels in cells and tissues and to characterize pharmacological properties of PARP inhibitors. In contrast to existing PAR-detection techniques, the LC-MS/MS method uses authentic isotope-labeled standards, which provide unequivocal chemical specificity to quantify cellular PAR in absolute terms with femtomol sensitivity. Using this platform we analyzed steady-state levels as well as stress-induced dynamics of poly(ADP-ribosyl)ation in a series of biological systems including cancer cell lines, mouse tissues, and primary human lymphocytes. Our results demonstrate a rapid and transient stress-induced increase in PAR levels by >100-fold in a dose- and time-dependent manner with significant differences between cell types and individual human lymphocyte donors. Furthermore, ex vivo pharmacodynamic studies in human lymphocytes provide new insight into pharmacological properties of clinically relevant PARP inhibitors. Finally, we adapted the LC-MS/MS method to quantify poly(ADP-ribosyl)ation in solid tissues and identified tissue-dependent associations between PARP1 expression and PAR levels in a series of different mouse organs. In conclusion, this study demonstrates that mass spectrometric quantification of cellular poly(ADP-ribosyl)ation has a wide range of applications in basic research as well as in drug development.

An automated Fpg-based FADU method for the detection of oxidative DNA lesions and screening of antioxidants.

The oxidation of guanine to 8-oxo-2'-deoxyguanosine (8-oxo-dG) is one of the most abundant and best studied oxidative DNA lesions and is commonly used as a biomarker for oxidative stress. Over the last decades, various methods for the detection of DNA oxidation products have been established and optimized. However, some of them lack sensitivity or are prone to artifact formation, while others are time-consuming, which hampers their application in screening approaches. In this study, we present a formamidopyrimidine glycosylase (Fpg)-based method to detect oxidative lesions in isolated DNA using a modified protocol of the automated version of the fluorimetric detection of alkaline DNA unwinding (FADU) method, initially developed for the measurement of DNA strand breaks (Moreno-Villanueva et al., 2009. BMC Biotechnol. 9, 39). The FADU-Fpg method was validated using a plasmid DNA model, mimicking mitochondrial DNA, and the results were correlated to 8-oxo-dG levels as measured by LC-MS/MS. The FADU-Fpg method can be applied to analyze the potential of compounds to induce DNA strand breaks and oxidative lesions, as exemplified here by treating plasmid DNA with the peroxynitrite-generating molecule Sin-1. Moreover, this method can be used to screen DNA-protective effects of antioxidant substances, as exemplified here for a small-molecule, i.e., uric acid, and a protein, i.e., manganese superoxide dismutase, both of which displayed a dose-dependent protection against the generation of oxidative DNA lesions. In conclusion, the automated FADU-Fpg method offers a rapid and reliable measurement for the detection of peroxynitrite-mediated DNA damage in a cell-free system, rendering it an ideal method for screening the DNA-protective effects of antioxidant compounds.

Quantitative analysis of WRN exonuclease activity by isotope dilution mass spectrometry.

Werner syndrome is a disorder characterized by a premature aging phenotype. The disease is caused by mutations in the WRN gene which encodes a DNA helicase/exonuclease which is involved in multiple aspects of DNA metabolism. Current methods mostly rely on radiometric techniques to assess WRN exonuclease activity. Here we present an alternative, quantitative approach based on non-radioactive isotope dilution mass spectrometry (LC-MS/MS). A oligoduplex substrate mimicking the telomeric sequence was used for method development. Released nucleotides, which correlate with the degree of oligoduplex degradation, were dephosphorylated, purified, and quantified by LC-MS/MS. Heavy-isotope-labeled internal standards were used to account for technical variability. The method was validated in terms of reproducibility, time-course and concentration-dependency of the reaction. As shown in this study, the LC-MS/MS method can assess exonuclease activity of WRN mutants, WRN's substrate and strand specificity, and modulatory effects of WRN interaction partners and posttranslational modifications. Moreover, it can be used to analyze the selectivity and processivity of WRN exonuclease and allows the screening of small molecules for WRN exonuclease inhibitors. Importantly, this approach can easily be adapted to study nucleases other than WRN. This is of general interest, because exonucleases are key players in DNA metabolism and aging mechanisms.

CE of tricyclic antidepressant clomipramine and metabolites: electromigration and wall adsorption.

CE of tricyclic antidepressants clomipramine and its metabolites demethylclomipramine, didemethylclomipramine and 8-hydroxyclomipramine resulted in partly extremely tailing peaks in bare fused-silica capillaries. Especially at high pH of the BGE this behavior was not unexpected as adsorption of the cationic analytes onto the negatively charged wall due to electrostatic attraction can be supposed. Less expected was the observation that peak tailing could not be overcome neither by using a capillary with dynamic coating with cationic CTAB added to the BGE, nor by the usage of a capillary permanently coated with polyvinyl alcohol (PVA), both operated at acidic pH. As this tailing was even more pronounced than with bare fused silica, and was suppressed upon addition of MeCN to the BGE, another source of adsorption than pure ion-ion interaction seems plausible. In the bare silica capillary the mobility, mu, of the analytes followed roughly the pH dependence of a monoacidic base, but two deviations from the sigmoid theoretical curve were evident: (i) even at low pH the mobilities were not constant; they decreased in contrary with pH over the entire range; (ii) the apparent pK(a) values of two analytes, derived at the pH with halve the mobility at low pH, are significantly smaller than the thermodynamic pK(a). Upon modifying the expression for mu = f(pH), and considering the pH dependence of the negative charge density at the wall by an additional term which takes chromatographic retention into account, an equation was derived which enables the description of the observed electromigration of the analytes as function of pH, pK(a) of analytes and surface silanol groups, actual mobility of analytes, distribution coefficient (or retention factor) due to adsorption including its pH dependence. The interplay of electrophoretic movement and residual adsorptive retention allowed to resolve the analytes finally in an uncoated capillary, namely at pH 7.65 (30 mM ionic strength), whereas at the cost of the robustness of the separation system.