Human Tail Research Articles

The human CRY1 tail controls circadian timing by regulating its association with CLOCK:BMAL1

Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.

Significant compaction of H4 histone tail upon charge neutralization by acetylation and its mimics, possible effects on chromatin structure

The intrinsically disordered, positively charged H4 histone tail is important for chromatin structure and function. We have explored conformational ensembles of human H4 tail in solution, with varying levels of charge neutralization via acetylation or amino-acid substitutions such as K→Q. We have employed an explicit water model shown recently to be well suited for simulations of intrinsically disordered proteins.Upon progressive neutralization of the H4, its radius of gyration decreases linearly with the tail charge q, the trend is explained using a simple polymer model. While the wild type state (q=+8) is essentially a random coil, hyper-acetylated H4 (q=+3) is virtually as compact and stable as a globular protein of the same number of amino-acids.Conformational ensembles of acetylated H4 match the corresponding K→X substitutions only approximately: based on the ensemble similarity, we propose K→M as a possible alternative to the commonly used K→Q.Possible effects of the H4 tail compaction on chromatin structure are discussed within a qualitative model in which the chromatin is highly heterogeneous, easily inter-converting between various structural forms. We predict that upon progressive charge neutralization of the H4 tail, the least compact sub-states of chromatin de-condense first, followed by de-condensation of more compact structures, e.g. those that harbor a high fraction of stacked di-nucleosomes. The predicted hierarchy of DNA accessibility increase upon progressive acetylation of H4 might be utilized by the cell for selective DNA accessibility control.

Intracellular pH controls WNT downstream of glycolysis in amniote embryos.

Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencingthe Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic β-catenin acetylation downstream of WNT signalling. As acetylated β-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.

Classification of the "human tail": Correlation between position, associated anomalies, and causes.

Numerous case reports have indicated that the "human tail" is not always a harmless protrusion but can be associated with anomalies such as occult dysraphic malformations. However, the definition and classification of this anomaly have not been discussed. A prevailing hypothesis is that the "human tail" is a residual embryonic tail. Herein, we attempted to classify and define the human tail and investigate the frequency of this anomaly. We first defined the human tail as a protrusion on the dorsal side of the lumbar, sacrococcygeal, and para-anal regions identified after birth. We collected case reports written in English, Japanese, French, German, and Italian that were published from the 1880s to the present. We discovered two important findings: (a) the cause of this anomaly may differ even though the "tails" resemble each other closely in appearance and (b) its position tends to be correlated with the type of anomaly and its associated cause. We propose a new classification of the human tail based on these findings. Our classification may facilitate more accurate treatment and precise case descriptions of the human tail.

CD300f immunoreceptor is associated with major depressive disorder and decreased microglial metabolic fitness

A role for microglia in neuropsychiatric diseases, including major depressive disorder (MDD), has been postulated. Regulation of microglial phenotype by immune receptors has become a central topic in many neurological conditions. We explored preclinical and clinical evidence for the role of the CD300f immune receptor in the fine regulation of microglial phenotype and its contribution to MDD. We found that a prevalent nonsynonymous single-nucleotide polymorphism (C/T, rs2034310) of the human CD300f receptor cytoplasmic tail inhibits the protein kinase C phosphorylation of a threonine and is associated with protection against MDD, mainly in women. Interestingly, CD300f-/- mice displayed several characteristic MDD traits such as augmented microglial numbers, increased interleukin 6 and interleukin 1 receptor antagonist messenger RNA, alterations in synaptic strength, and noradrenaline-dependent and persistent depressive-like and anhedonic behaviors in females. This behavioral phenotype could be potentiated inducing the lipopolysaccharide depression model. RNA sequencing and biochemical studies revealed an association with impaired microglial metabolic fitness. In conclusion, we report a clear association that links the function of the CD300f immune receptor with MDD in humans, depressive-like and anhedonic behaviors in female mice, and altered microglial metabolic reprogramming.

Posttranslational lysine 2-hydroxyisobutyrylation of human sperm tail proteins affects motility.

Does lysine 2-hydroxyisobutyrylation, a newly identified protein posttranslational modification (PTM), occur in human sperm and affect human sperm function? Lysine 2-hydroxyisobutyrylation mainly occurs in human sperm tail proteins, and excessive lysine 2-hydroxyisobutyrylation affects human sperm motility. PTM is regarded as an important pathway in regulating sperm function since mature sperm are almost transcriptionally silent. However, only phosphorylation was extensively studied in mature sperm to date. Lysine 2-hydroxyisobutyrylation, a newly characterised PTM, is broadly conserved in both eukaryotic and prokaryotic cells. Although histone lysine 2-hydroxyisobutyrylation has been shown to be associated with active gene expression in spermatogenic cells, the presence, regulatory elements and function of lysine 2-hydroxyisobutyrylation have not been characterised in mature sperm. Sperm samples were obtained from normozoospermic men and asthenozoospermic men who visited the reproductive medical centre at Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, China, between May 2017 and November 2018. In total, 58 normozoospermic men and 65 asthenozoospermic men were recruited to participate in this study. Lysine 2-hydroxyisobutyrylation was examined using immunoblotting and immunofluorescence assays using a previously qualified pan anti-lysine 2-hydroxyisobutyrylation antibody. The immunofluorescence assay was imaged using super-resolution structured illumination microscopy. Sperm viability was examined by using the eosin staining method, and sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm penetration ability was determined by evaluating the ability of the sperm to penetrate a 1% (w/v) methylcellulose solution. The level of intracellular adenosine triphosphate (ATP) was detected using a rapid bioluminescent ATP assay kit. Lysine 2-hydroxyisobutyrylation was present in several proteins (20-100kDa) mainly located in the tail of human sperm. Sperm lysine 2-hydroxyisobutyrylation was derived from 2-hydroxyisobutyrate (2-Hib) and was regulated by acyltransferase P300 and nicotinamide adenine dinucleotide-dependent lysine deacylase sirtuins. Elevation of sperm lysine 2-hydroxyisobutyrylation by 2-Hib decreased total motility, progressive motility, penetration ability and ATP level of human sperm. Interestingly, the level of sperm lysine 2-hydroxyisobutyrylation was higher in asthenozoospermic men than that in normozoospermic men and was negatively correlated with the progressive motility of human sperm. Furthermore, high levels of lysine 2-hydroxyisobutyrylation in asthenozoospermic men accompanied decreased ATP levels. Although the present study indicated the involvement of sperm lysine 2-hydroxyisobutyrylation in regulating human sperm motility, the underlying mechanism needs to be further illustrated. The findings of this study provide insight into the novel role of lysine 2-hydroxyisobutyrylation in human sperm and suggest that abnormality of sperm lysine 2-hydroxyisobutyrylation may be one of the causes for asthenozoospermia. National Natural Science Foundation of China (81771644 to T.L. and 81871207 to H.C.); Natural Science Foundation of Jiangxi province (20171ACB21006). The authors have no conflicts of interest to declare.

Novel Expression of GABAA Receptors on Resistance Arteries That Modulate Myogenic Tone

The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABA_A receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABA_A ligands. We questioned whether GABA_A receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABA_A subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABA_A receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABA_A receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABA_A receptors in the modulation of myogenic tone in mouse and human resistance arteries.

Human tail- a rare anatomical mystery: A case report

Background: Human tails are rare entities and can be classified as true tails and pseudotails. True tails are composed of adipose tissue, connective tissue, muscles, vessels, nerves and mechanoreceptors whereas pseudotails may be an anomalous prolongation of the coccygeal vertebra, lipoma, teratoma, chondrodystrophy or parasitic fetus. Case Report: A 3-month-old male baby had a tail like appendage since birth which was gradually increasing in size. After complete workup, an elective resection was done under general anesthesia. Postoperative recovery was uneventful. Conclusion: Surgical excision of the tail is the ultimate and may be the only surgical intervention required in these patients.

Amniotic band associated with human tail-like cutaneous appendage and spinal dermoid tumor: An exceedingly rare triad

Introduction: The association between amniotic band syndrome (ABS) and spinal dysraphism is an extremely rare entity that was paucity reported in the literature so far and similar conditions such multiple asymmetric encephaloceles and anencephaly were also previously described with ABS. Methods: In this case report, we described a male newborn in whom ABS was associated with human tail and lumbar dysraphism. A surgical approach was performed with intraoperative neurophysiological monitoring in which the tail and amniotic band were en bloc resected with favorable outcome. Dermoid tumor was identified by histopathological analysis Conclusion: The occurrence of spinal dysraphism combined with ABS should be managed carefully in order to avoid spinal cord damage. provides very good results for a number of indications in children. Every effort should be made to optimize the selection of surgical candidates on the basis of their clinical features.

LAX28 is required for the stable assembly of the inner dynein arm f complex, and the tether and tether head complex in Leishmania flagella.

Motile eukaryotic flagella beat through coordinated activity of dynein motor proteins; however, the mechanisms of dynein coordination and regulation are incompletely understood. The inner dynein arm (IDA) f complex (also known as the I1 complex), and the tether and tether head (T/TH) complex are thought to be key regulators of dynein action but, unlike the IDA f complex, T/TH proteins remain poorly characterised. Here, we characterised T/TH-associated proteins in the protist Leishmania mexicana Proteome analysis of axonemes from null mutants for the CFAP44 T/TH protein showed that they lacked the IDA f protein IC140 and a novel 28-kDa axonemal protein, LAX28. Sequence analysis identified similarities between LAX28 and the uncharacterised human sperm tail protein TEX47, both sharing features with sensory BLUF-domain-containing proteins. Leishmania lacking LAX28, CFAP44 or IC140 retained some motility, albeit with reduced swimming speed and directionality and a propensity for flagellar curling. Expression of tagged proteins in different null mutant backgrounds showed that the axonemal localisation of LAX28 requires CFAP44 and IC140, and the axonemal localisations of CFAP44 and IC140 both depend on LAX28. These data demonstrate a role for LAX28 in motility and show mutual dependencies of IDA f and T/TH-associated proteins for axonemal assembly in Leishmania.

Human tails: Interdisciplinary treatment for dorsal cutaneous appendages and associated spectrum of spinal dysraphism

The dorsal cutaneous appendage, or human tail, is a rare congenital anomaly that can span a broad spectrum of tissue and spinal involvement. Associated occult spinal anomalies may not be limited to the lumbar region and can occur at any point along the length of the spinal column. Five cases of human tails are presented with varying degrees of soft tissue anomalies, spinal dysraphism, and neurologic symptoms. Patients range in age from newborn to 22 years. The spectrum of pathology includes those isolated to skin and subcutaneous tissue as well as underlying spinal dysraphism and tethered cord. Surgical treatment ranged from simple tail excisions to microsurgical debulking and spinal cord detethering. The clinical presentation of a lumbosacral appendage should prompt the workup for underlying spinal anomalies at all levels of the spinal cord. Without treatment, the neurologic sequelae of occult spinal dysraphism can be progressive. With proper preoperative evaluation and interdisciplinary treatment, the soft tissue anomaly as well as the spinal dysraphism can be surgically treated to restore function or prevent further neurologic compromise.

Soft liquid metal nanoparticles achieve reduced crystal nucleation and ultrarapid rewarming for human bone marrow stromal cell and blood vessel cryopreservation

High warming rates during cryopreservation are crucial and essential for successful vitrification. However, realizing a faster warming rate in low-concentration cryoprotective agents appears to be challenging for conventional warming process through convective heat transfer. Herein, we developed a liquid metal (LM) nanosystem that can act as a spatial source to significantly enhance the warming rates with near-infrared laser irradiation during the warming process. The synthetic Pluronic F127-liquid metal nanoparticles (PLM NPs) displayed multiple performances with uniform particle size, superior photothermal conversion efficiency (52%), repeatable photothermal stability, and low cytotoxicity. Particularly, it is more difficult for the liquid PLM NPs with less surface free energy to form crystal nucleation than other solid NPs such as gold and Fe3O4, which is beneficial for the cooling process during cryopreservation. The viability of human bone marrow-derived mesenchymal stem cells postcryopreservation reached 78±3%, which is threefold higher than that obtained by the conventional warming method (25±6%). Additionally, the cells postcryopreservation maintained their normal attachment, proliferation, surface marker expression, and intact multilineage differentiation properties. Moreover, the results of mouse tails including blood vessel cryopreservation showed a relatively improved intact structure when using PLM NP rewarming compared with the results of conventional warming. The new LM nanosystem provides a universal platform for cryopreservation that is expected to have potential for widespread applications including bioengineering, cell-based medicine, and clinical translation. Statement of significanceIn this study, we fabricated soft liquid metal nanoparticles with high photothermal conversion efficiency, repeatable photothermal stability, and low cytotoxicity. Particularly, soft liquid metal nanoparticles with less surface free energy and suppression effects of ice formation were first introduced to mediate cryopreservation. Superior ice-crystallization inhibition is achieved as a result of less crystal nucleation and ultrarapid rewarming during the freezing and warming processes of cryopreservation, respectively. Collectively, cryopreservation of human bone marrow stromal cells (HBMSCs) and mouse tails including blood vessels can be successfully performed using this new nanoplatform, showing great potential in the application of soft nanoparticles in cryopreservation.

Human myosin 1e tail but not motor domain replaces fission yeast Myo1 domains to support myosin-I function during endocytosis

In both unicellular and multicellular organisms, long-tailed class I myosins function in clathrin-mediated endocytosis. Myosin 1e (Myo1e) in vertebrates and Myo1 in fission yeast have similar domain organization, yet whether these proteins or their individual protein domains are functionally interchangeable remains unknown. In an effort to assess functional conservation of class I myosins, we tested whether human Myo1e could replace Myo1 in fission yeast Schizosaccharomyces pombe and found that it was unable to substitute for yeast Myo1. To determine if any individual protein domain is responsible for the inability of Myo1e to function in yeast, we created human-yeast myosin-I chimeras. By functionally testing these chimeric myosins in vivo, we concluded that the Myo1e motor domain is unable to function in yeast, even when combined with the yeast Myo1 tail and a full complement of yeast regulatory light chains. Conversely, the Myo1e tail, when attached to the yeast Myo1 motor domain, supports localization to endocytic actin patches and partially rescues the endocytosis defect in myo1Δ cells. Further dissection showed that both the TH1 and TH2-SH3 domains in the human Myo1e tail are required for localization and function of chimeric myosin-I at endocytic sites. Overall, this study provides insights into the role of individual myosin-I domains, expands the utility of fission yeast as a simple model system to study the effects of disease-associated MYO1E mutations, and supports a model of co-evolution between a myosin motor and its actin track.

Human Rad17 C-terminal tail is phosphorylated by concerted action of CK1δ/ε and CK2 to promote interaction with the 9–1–1 complex

The ATR–dependent DNA damage checkpoint is one of the major checkpoint pathways. The interaction between the Rad17–RFC2-5 and 9–1–1 complexes is central to the ATR–Chk1 pathway. However, little is known about the regulation of the interaction. We recently showed that vertebrate Rad17 proteins share a conserved C-terminal tail and that the C-terminal tails have a conserved amino acid motif named iVERGE that must be intact for the interaction between Rad17 and the 9‒1‒1 complex. In human Rad17, the Y665 and S667 residues are conserved in iVERGE. The Rad17-S667 residue is phosphorylated by CK2, and the phosphorylation is important for the interaction with the 9‒1‒1 complex. Here, we show that a C-terminal threonine residue of Rad17, T670 in human Rad17, is constitutively phosphorylated in vivo. The T670 phosphorylation is important for the S667 phosphorylation, and vice versa. Phosphomimetic mutations in the T670 residue promote the interaction with the 9–1–1 complex. The T670 and Y665 residues show functional redundancy, and their roles are dependent on the S667 phosphorylation. Rad17-T670 is phosphorylated by casein kinase 1δ/ε. Our data suggest that iVERGE integrates multiple signaling pathways to regulate the ATR–Chk1 pathway.

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail.

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane.

Clustering of integrin \u03b2 cytoplasmic domains triggers nascent adhesion formation and reveals a protozoan origin of the integrin-talin interaction

Integrins and integrin-dependent cell-matrix adhesions are essential for a number of physiological processes. Integrin function is tightly regulated via binding of cytoplasmic proteins to integrin intracellular domains. Yet, the complexity of cell-matrix adhesions in mammals, with more than 150 core adhesome proteins, complicates the analysis of integrin-associated protein complexes. Interestingly, the evolutionary origin of integrins dates back before the transition from unicellular life to complex multicellular animals. Though unicellular relatives of metazoa have a less complex adhesome, nothing is known about the initial steps of integrin activation and adhesion complex assembly in protozoa. Therefore, we developed a minimal, microscope-based system using chimeric integrins to investigate receptor-proximal events during focal adhesion assembly. Clustering of the human integrin β1 tail led to recruitment of talin, kindlin, and paxillin and mutation of the known talin binding site abolished recruitment of this protein. Proteins indirectly linked to integrins, such as vinculin, migfilin, p130CAS, or zyxin were not enriched around the integrin β1 tail. With the exception of integrin β4 and integrin β8, the cytoplasmic domains of all human integrin β subunits supported talin binding. Likewise, the cytoplasmic domains of integrin β subunits expressed by the protozoan Capsaspora owczarzaki readily recruited talin and this interaction was based on an evolutionary conserved NPXY/F amino acid motif. The results we present here validate the use of our novel microscopic assay to uncover details of integrin-based protein-protein interactions in a cellular context and suggest that talin binding to integrin β cytoplasmic tails is an ancient feature of integrin regulation.

Human Tail: A Benign Condition Hidden Out of Social Stigma and Shame in Young Adult - A Case Report and Review.

A human tail is a rare congenital anomaly which mostly presents immediately after birth or in early childhood. Here, we are presenting a case of 17-year-old male who presented with 18-cm long tail, which was hidden till this age because of social stigma and shame. This is longest human tail reported of our knowledge. This patient presented to us because he started having difficulty, pain while sitting, and discomfort in daily activities because of long tail. We suspect there are far more cases of human tails in developing world which are hidden because of lack of knowledge, illiteracy, social stigma, and shame. This patient had no neurological deficit and had no bowel/bladder involvement. The radiological investigation was suggestive of spina bifida at L5 and S3–S4 levels (site of attachment of the tail), and tip of the coccyx had bony spur directed posteriorly. Intraoperatively, no connection was found between tail and neural tissue. Human tail is associated with occult spinal lesions in 50% of cases, hence careful neurological and radiological investigations are warranted before surgical exploration.

Quantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrix

By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 µm. Correlation of the secondary electron and the hyperspectral images proved ~40 nm resolution in the optical channel, obtained due to a short carrier diffusion length, suppressed by fibril dimensions and poor electrical conductivity specific to their organic composition. By correlating spectrally analyzed auto-cathodoluminescence with mass spectroscopy data, we differentiated spectral signatures of two extracellular matrices, namely human fibrin complex and rat tail collagen isolate, and uncovered differences in protein distributions of isolated extracellular matrix networks of heterogeneous populations. Furthermore, we demonstrated that cathodoluminescence can monitor the progress of a human cell-mediated remodeling process, where human collagenous matrix was deposited within a rat collagenous matrix. The revealed change of the heterogeneous biological composition was confirmed by mass spectroscopy.

Human H4 tail stimulates HIV-1 integration through binding to the carboxy-terminal domain of integrase

The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)•viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.

A technique for separating viable islets of Langerhans from a fragment of human pancreatic tail

Introduction.Modern techniques of tissue engineering in the treatment of some degenerative diseases suggest the prospective viability of the biomedical technologies based on the creation of the equivalent of the damaged tissue (organ), including the tissue-engineered construct (TEC) of the endocrine pancreas (EP). Obtaining viable islets of Langerhans (IL) from the pancreas is a decisive step towards the creation of a TEC EP. The classic method of IL separation is based on enzymatic digestion of pancreatic tissue and further islet purification in ficoll density gradient during centrifugation, which adversely affects the morphofunctional state of IL.The aimof the study was the development of a method for separating viable pancreatic islets from a fragment of human pancreatic tail with different cold ischemia times.Materials and methods.A procedure of IL separation is proposed to be conducted without the use of EP tissue collagenase perfusion in the Ricordi chamber at the stage of IL separation and without ficoll solution with a varying density gradient at the stage of IL purification. Identification of IL obtained was performed by dithizone staining. The IL viability was evaluated using the LIVE/DEAD ® Cell Viability Kit. Histological analysis of the initial material included routine staining methods as well as immunohistochemical staining of the main types of islet cells.Results.The morphological study of the EP fragments at different times of cold ischemia did not reveal significant differences in the histological presentation of the organ parenchyma; the islet structure appeared intact. Vital staining confirmed the separated IL viability in vitro for at least 1–3 days.Conclusion.The proposed method of pancreatic tissue treatment allowed to reduce the number of stages, thereby minimizing the adverse effects of centrifugation and ficoll on the integrity of IL. It is possible to obtain the necessary amount of viable IL from a small EP fragment with the cold ischemia time of up to 19 hours, which can be used to create a TEC of a pancreas.