Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including  Scorpions, a characteristic group of arthropods, diverged from other arachnids relatively early, at about 440 million years ago ,2. TheirMorphological phylogenetic analyses suggest that scorpions are sister taxa to either the rest of the arachnids or to the Opiliones . HoweverAmong the 18 scorpion families with more than 2200 species described in the world, about thirty species have been identified as potentially deadly toxic to humans .Our studies on scorpions are based on two remarkable aspects of this group: Their evolutionary origin and systematic classification, and the diversity and origin of their venom components, with emphasis on the use of these components as potential sources of molecules with therapeutic applications .Scorpions have fascinated scientists and laypersons for their venom, which is a complex mixture of bioactive components secreted in specialized organs . These aHere, we review the diversity of scorpions in Iran. We also highlight the importance of the venomic studies of Iranian scorpions. Establishing what is known about of scorpions and scorpion venom in Iran will allow the identification of important gaps to be addressed in the future.Iran is a vast land with diverse climates. There are two main mountain ranges in Iran: (1) Alborz; and (2) Zagros and neighboring mountains. Diverse climates in Iran are the direct result of the presence of the Persian Gulf, the Oman Sea bordering Southeastern Iran, and the Caspian Sea in Northern Iran. According to Safaei-Mahroo et al.  16 terreIran is located in a strategic position in the Palearctic region and is a bridge between the Oriental and African zoogeographical regions, suggesting the possibility of endemic arthropod species in this region. Among these arthropods, scorpions stand out as there are diverse species in desert and semi-desert regions of Iran ) [th century, Max Vachon carried out preliminary studies on the scorpions of Iran, with a later report of two families, nine genera, and 15 species [Androctonus baluchicus, Androctonus crassicauda, and Androctonus finitimus in Iran. However, Yağmur et al. [Androctonus crassicauda and Androctonus robustus are present in Iran while rejecting the occurrence of Androctonus baluchicus and Androctonus finitimus in Iran. Compsobuthus kafkai and Compsobuthus sobotniki were synonymized with Sassanidotus gracilis [Hottentotta alticola alticola are present in Iran but Mirshamsi et al. [Hottentotta alticola alticola are in doubt. Mirshamsi [Mesobuthus phillipsi includes the Mesobuthus phillipsi pachysoma and Mesobuthus phillipsi mesopotamicus subspecies. Although Vachon [Odontobuthus odonturus in Iran, Lowe [Odontobuthus odonturus in Iran. Based on field work, study collections, literature reviews, and personal communications, the total number of species confirmed within the Iranian border is 78 species and subspecies belonging to 19 genera and four families. The family Buthidae is the most diverse with 68 species and subspecies (87.17%), followed by Hemiscorpiidae with seven species (8.97%), Scorpionidae with two subspecies (2.56%), and Diplocentridae with one species (1.28%) [Historically, the fauna of Iran has been studied by many researchers. The earliest descriptions of the first species were made by Olivier , 1807)) . Later, , 1807)) ,17,18,1907) . Lat, 1807)) ,24,25,26, 1807))  and Wern, 1807))  describe species . Later,  species  reported species ,32 repor species  reported species ,35,36,37 species  reported species  and Mirs species , there ar et al.  believedgracilis . Farzanpi et al.  believedirshamsi  believedh Vachon , Farzanph Vachon , and Nejh Vachon , reportean, Lowe  rejected (1.28%) ,47,48,49Animal venoms are a mixture of different compounds for defense and prey capture. Many peptide toxins from deadly animal venoms have been influenced by ion channel  functions. The ion channels play important roles in the regulation of the heart beat and neuronal excitability ,51.Scorpiops tibetanus. This peptide has immunosuppressive and anti-inflammatory effects that suggest its potential use as a new peptide medicine for human diseases [Scorpion venoms are certainly important natural drug resources for medical applications. In scorpions, family Buthidae has always been interesting from the public health perspective in terms of their dangerous venoms. Many studies have concentrated on non-Buthidae families and reported several new venom peptides and proteins which have shown unique primary structures and biological activities ,53,54,55diseases .Scorpion toxins have been used in variety of fields, including biotechnology (examining the effects on ion channels), identifying cancer mass , treatinPandinus imperator and Scorpio maurus palmatus have peptides named imperatoxin A (IpTxa) and maurocalcin (MCa), respectively, and these venoms are of interest in many cardiovascular diseases [The venoms of diseases ,63.+, Na+, Ca2+ and Cl_), peptide length , structural scaffold α, αβ and βαββ), disulfide bonds (three or four and pairing pattern), the mechanism of action/binding sites (α- or β-like toxins), their cellular target, and others. [Classification of polypeptide toxins is important for understanding the structure–function relationship of each individual group. The major criteria used for classification are based on receptor/ion channel specificity  and hyaluronidases, proteins, and peptides  [Biologically, venoms of scorpions are diverse and have activity due to their predatory and defensive use in nature ,70. In ahannels) ,72,73.Several peptide toxins in venomous animals are being considered for pharmacological applications, including treating pain, diabetes, multiple sclerosis, and cardiovascular illnesses ,51,74.Worldwide, peptides are progressively emerging as a novel class of therapeutics. In total, 438 peptides are represented in the pharmaceutical trade, including 72 in Phase III clinical trials and 48 that have been approved. Four are famous and sold in the pharmaceutical market: Copaxane, Lupron, Byetta, and Forteo . The majSeveral studies have reported that scorpion peptides have antineoplastic activity ,77. SomeAnti-proliferative, cytotoxic, and apoptogenic properties of scorpion venom peptides on different types of cancers have also been discovered. Scorpion venom peptides with fluorescent labeling have been used to visualize the boundaries of cancerous tissues in cancer patients .Androctonus crassicauda (100 μg/mL) blocked propagation of MCF-7 cells by suppressing S-phase of the cell cycle [For example, the venom of ll cycle ,80,81. Hll cycle .Odontobuthus doriae causes apoptosis in breast cancer by depolarizing mitochondria membranes and controlling S-phase proliferation in human breast cancer cells. MCF-7, on the other hand, reduced catalase activity, glutathione production, DNA fragmentation, and apoptosis [The venom of poptosis .Ion channels include voltage-gated sodium, calcium, and potassium channels which create electrical signals required for action potential generation and conduction, and are the molecular targets for a broad range of potent neurotoxins .vs) are composed of transmembrane proteins that conduct sodium ion (Na+) into the cytosol upon activation. An Nav contains a main α-subunit (220–260 kDa) and one or two auxiliary β-subunits (30–40 kDa) [+ selectivity [vs play an important role for action potential (AP) generation and proliferation in excitable cells, including cardiac myocytes, skeletal muscle cells, and neurons [vs are also marginally expressed in non-excitable cells involved in noncanonical roles in controlling some pathophysiological activities [Voltage-gated sodium channels (Na–40 kDa) ,84. The ectivity . Navs pl neurons ,87. Navstivities .v subtypes  in nociceptive transduction. These Navs probably show attractive targets for analgesic drug discovery. However, their channels also introduce valuable probes to demonstrate the structures, gating properties, and cellular functions of ion channels  Pore-blocking toxins that inhibit Na+ conductance by interacting with neurotoxin site 1, including tetrodotoxin (TTX), μ-conotoxins, and saxitoxin (STX); (2) toxins that negatively shift the activation voltage and produce a persistent activation by connecting to membrane-embedded neurotoxin site 2 , or 5 (such as ciguatoxin and brevetoxin), and that prefer to interact with the open state of channel; and (3) toxins that delay inactivation by binding to extracellular neurotoxin site 3, such as sea-anemone toxins and α-scorpion toxins. According to recent investigations, β-scorpion toxins shift activation voltage to either a depolarized or hyperpolarized direction through main effects of neurotoxin site 4. Delta-conotoxin prolongs channel inactivation, similarly to that caused by α-scorpion toxins, by binding to neurotoxin site 6. Although the neurotoxin binding sites are topologically distinct, allosteric coupling has been elucidated between sites 3 and 6 and between sites 2 and 5 [1B-4B genes) have been reported in mammals. The β-subunits are type I transmembrane proteins, including an extracellular signal peptide in the N-terminus, a transmembrane segment, and an immunoglobulin domain [v α-subunits [Three categories of Na 2 and 5 ,91. In an domain ,93. Bothsubunits ,91,93,94V channels [There are several studies that provide evidence of these mechanisms. As described above, there are several kinds of toxins in scorpion venoms that modulate the activity of ion channels, and these are usually responsible for several signs of envenoming. From the human perspective, the most medically important toxins are those that modulate mammalian Nachannels ,69.Odontobuthus doriae) was OD1, which blocks the fast inactivation of mammalian channels Nav1.7, Nav1.4, and Nav1.6 [1 inhibits fast inactivation of the para/tipE insect channel (EC50 80 nM), but it scarcely influences mammalian Nav1.3 or Nav1.5 (EC50 > 1 μM) and has no effect on Nav1.2 and Nav1.8. OD1 rapidly induces pain when injected into animals in association with, or without, veratridine, and has been used to test the analgesic effects of Nav1.7 blockers in vivo [+ currents resulting in incomplete inactivation in steady-state conditions has been demonstrated by OD1 [+ current causes sustained depolarization of the cell membrane, and the remaining Nav1.7 channels that were not affected by OD1 are trapped in the inactivated state, resulting in the loss of electrical excitability of nociceptor neurons [The first neurotoxin targeting voltage-gated sodium channels extracted from an Iranian scorpion venom . Thus far, 293 KTxs have been described in UniProt and 174 KTxs have been described in the Kalium database at http://kaliumdb.org/ [v1, Kv3, Kv4, Kv7, Kv11, and KCa channels [Scorpion venom has Kmdb.org/ ,111. Thechannels .+ channel specific toxins (KTx), and are important tools for the structural and functional characterization of various K+ [v channel inhibitors have medical applications in the treatment of various specific human diseases, especially autoimmune disorders, inflammatory neuropathies, and cancer [Venom of scorpions are an important source of Krious K+ ,114. Kv d cancer .V1.3 channels contribute to the control of calcium signaling to induce T-cell proliferation, immune activation, and cytokine production. In many autoimmune diseases, effector memory T (TEM) cells, which play major roles, are controlled by blocking KV1.3 channels on the membrane. Animal toxins are capable of suppressing the activation and proliferation of TEM cells and may improve TEM cell-mediated autoimmune diseases, for example in multiple sclerosis and type I diabetes mellitus [Autoimmune diseases are usually accompanied by tissue injury caused by autoantigen-specific T-cells. Kmellitus .+ channel toxin isolated from the venom of the Iranian scorpion Odontobuthus doriae (OdK1) was categorized as α-KTx 8.5. The pharmacological effects of OdK1 were investigated on Xenopus laevis oocytes heterologously expressing Kv1.2 channels. OdK1 selectively blocked the currents through Kv1.2 channels, with no effect on the other channels tested [The first Ks tested .Odonthobuthus doriae, one of the endemic scorpion species of Iran. OdK2 is composed of 38 amino acid residues, including six conserved half-cystine residues and a C-terminal lysine residue. The toxin was named KTX3.11. Pharmacologically, OdK2 selectively suppresses the currents via Kv1.3 channels. [There is a Kv1.3 channel-selective toxin, OdK2, in venom of hannels. .+ channel blocker, was isolated from the venom of Hemiscorpius lepturus, which is classified in subfamily six of the α-KTx family of potassium channel scorpion toxins and has the highest amino acid sequence similarity with maurotoxin (MTX), extracted from the Tunisian scorpion Scorpio maurus palmatus. Additionally, MTX is also a K+ channel inhibitor with 34 (instead of 35) amino acid residues.Hemitoxin (HTx), a Kv1.1, Kv1.2, and Kv1.3 channels. HTX has subtype-selective effects on K+ channels. It is 20 times less potent on Kv1.2 channels, and 90 times more potent on Kv1.3 channels, compared to the α-KTx6 family member MTX [HTX reversibly inhibits type Kmber MTX .v) play a critical role in electrical signaling, converting depolarization of the cell membrane to an influx of calcium ions that initiates contraction, secretion, neurotransmission, and other intracellular regulatory events [v) facilitate cellular calcium influx in response to membrane depolarization. They control hormone secretion, neurotransmitter release, propagation of cardiac action potential, muscle contraction, and gene expression in different cell types [v channels, the α1 subunit of Cav channels is organized in four homologous domains (I–IV), each containing six TM segments (S1–S6). The S1–S4 segments are involved in the voltage sensor, whereas S5–S6 constitutes the pore. Auxiliary subunits usually associate with α1, regulating channel expression and function. Cav channels are classified in the following groups in terms of electrophysiological and pharmacological properties and tissue distribution—L-type (Cav1 subfamily: Cav1.1-Cav1.4); P/Q-, N-, and R-types ; and T-type (Cav3 subfamily: Cav3.1-Cav3.3) [Voltage-gated calcium channels (Cay events . Voltagell types . Similar-Cav3.3) .2+ channels of the sarcoplasmic reticulum in muscle and of the endoplasmic reticulum in other cells. In striated muscle fibers, contraction has been carried out via RyRs that releases Ca2+ rapidly. There are various isoforms of ryanodine-sensitive calcium channels including RyR1, RyR2, and RyR3. In mammals, RyR1 is expressed mainly in skeletal muscle and RyR2 is expressed mainly in cardiac muscle. RyR3 seems to be confined to the brain, smooth muscle, and epithelial cells. RyR1 and RyR2 are expressed in some of these tissues as well. The activity of surface-membrane Ca2+ channels, the dihydropyridine receptors (DHPRs), stimulates the activation of RyRs during the early part of the excitation–contraction linking cascade [The various subfamilies have been presented for classification of calcium channels as follows: Voltage-gated channels, voltage independent channels, and ligand-activated channels. Ligand-activated channels include the ryanodine receptors (RyRs), which are high-permeability Ca cascade ,122.Hadrurus gertschi [There is selective activity on ryanodine receptors (RyRs) by the venom of gertschi .Odontobuthus doriae, one calcium channel toxin was recognized and is referred to as ODCaTx1 (ID: KU365856) [Hottentotta judaicus [In U365856) . It sharjudaicus . Analysijudaicus .Hemiscorpius lepturus, it represents 0.6% of the total protein content [Hemicalcin is a new toxin that was extracted from the venom of  content .− channels are classifed into three groups: Ligand-gated Cl− channels, the cystic fibrosis transmembrane conductance regulator (CFTR) channel, and the CLC channels [−/H+ antiporters that are thought to contribute to plasma membrane transport, lysosomal acidification, and the maintenance of the cell membrane potential [CLC chloride channels contain one pore per subunit (a ‘double-barreled’ channel), and also provide clues about gating and permeation . Cl− chachannels . The mamotential ,129,130.Short scorpion toxin chloride channel inhibitors are short-chain neurotoxins (SCNs) that block small-conductance chloride channels. They are 30-40-residue long and contain four intramolecular disulphide bridges, which have been labeled as C1-C4, C2-C6, C3-C7, and C5-C8 ,132,133.Odontobuthus doriae venom (ID: KU-365857); it is composed of four intramolecular disulfide bridges and a putative conserved domain belonging to the toxin-5 superfamily. There are a variety of secreted short scorpion toxins in the superfamily. Such toxins are not associated with the toxin-2 superfamily (pfam00451) that affects potassium channels [According to Naderi Soorki et al. , one chlchannels .These days, antimicrobial components represent one of the important resources for modern medical care . Natural2, and Kn2-7) showed powerful anti-HIV activity [Globally, approximately 36.7 million people suffer HIV, but still there is no definite cure to eradicate HIV transmission . The fivactivity .Moreover, some distinctive characteristics of scorpion physiology are related to the mixture, and to individual effects. These properties are mostly found in the Buthidae family, whose venom is pharmacologically the most important and prominent compared to non-buthids. Hemocyanin is a protein for transporting oxygen in all scorpions and has the ability to bind with molecular oxygen in reverse. The protein has three parts with an enzymatic role: Pseudo-catalase, peroxidase, and superoxide-dismutase ,140,141.Hemiscorpius lepturus scorpion venom contains several components with significant anti-HIV activity, suggesting it as a potential source of novel therapeutic agents against HIV infection [nfection .Mesobuthus eupeus venom glands was isolated that was encoded by a 213 bp cDNA fragment. The full-length sequence of the coding region was 210 bp and included an open reading frame of 70 amino acid residues with a predicted molecular mass of 7970.48 Da and theoretical pI of 9.10. The precursor (70 amino acid residues) includes a signal peptide of 23 amino acid residues and a mature peptide of 34 amino acid residues with no disulfide bridge. The resulted peptide of Mesobuthus eupeus has been named MeVAMP-2 (98%), MeVAMP-9 (60%). The other AMPs have been reported from Mesobuthus martensii (94%) and Buthus occitanus israelis (82%) [An antimicrobial toxin extracted from is (82%) .Odontobuthus doriae, called ODAMP1-6. Four of them  have signal peptides with peptides containing 55, 51, 52, and 51 amino acid residues, respectively. There are no signal peptides in ODAMPs . ODAMP1 (ID: KU212812) has 78 amino acid residues and resembles the antimicrobial peptide androcin 18-1 from Androctonus bicolor scorpion (78% identity). ODAMP6 (ID: KU365855) is a short chain peptide with only 47 amino acid residues, which is similar to Tx65 with antimicrobial activity from Buthus occitanus israelis (100% identity), and which could be beneficial and applicable in drug and food industries [There are six AMPs in venom of dustries .Odontobuthus doriae include ODVP4; KU365871 [Hemiscorpius lepturus include HLMP1; KX924496, HLMP2; KX924497, HLMP3; KX924498 [Matrix metalloproteinases (MMPs) affect cellular activities like growth and cell differentiation directly or indirectly ,146,147.KU365871  and thosKX924498 ,152,153.1, A2, C, and D) are a type of enzyme with a high disulfide bridge content and a conserved histidine/aspartic catalytic dyad. They that act on phospholipids to produce different products, including lysophospholipids, diacylglycerols (DGs), free fatty acids (FFAs), choline phosphate, and phosphatidates [2 (PLA2) include secreted (sPLA2), cytosolic (cPLA2), calcium independent (iPLA2), and platelet activating factor acetyl hydrolase (PAF-AH) or lipoprotein PLA2 (LpPLA2). In vertebrates and invertebrates, the most common type of PLA is sPLA2, which is grouped into 15 types [Phospholipases (PLA)  is the most common type of phospholipase observed in animals including vertebrates and invertebrates. According to primary sequences alignments, disulfide bond patterna, and their biochemical properties, it is divided into 15 groups ,159. TheHeterometrus fulvipes [Mesobuthus tamulus [Pandinus imperator [Anuroctonus phaiodactylus [Heterometrus laoticus [Hemiscorpius lepturus [Scorpio maurus [Hemiscorpius lepturus has potent phospholipase D activities that have been related to the highly toxic  necrosis activity of the venom [sPLA2 of scorpion venom are considered as group III and contained a long enzymatic chain and a short covalently fixed C-terminal chain generated after the release of five residues (penta-peptide) during the maturation processes . HfPLA2 fulvipes , MtPLA2  tamulus , Imperat tamulus  and Phosmperator , Phaiodadactylus , Heteromlaoticus , Hemiliplepturus , and Sm-o maurus . Hemiscohe venom .2 named hemilipin was recently isolated from dangerous scorpion of Iran, Hemiscorpius lepturus [2 activity on egg yolk phospholipids. Hemilipin widely affects angiogenesis in vitro and in vivo, whereas it doesn’t have any effect on apoptosis. Additionally, the study demonstrated that this new non-toxic sPLA2 could be used as an innovative tool to disrupt human angiogenesis at various points [2: Hemilipin2. This component has a robust calcium-dependent PLA2 activity and influences angiogenesis without any cytotoxic or apoptotic effects both in vitro and in vivo. However, there is a prominent capability in hemilipin2 to prevent blood vessel formation both in vitro and in vivo. The results suggest a beginning point to produce novel molecules that act as specific suppressors of human angiogenesis.In addition, a novel sPLAlepturus . Edman ds points . In a sus points  proposedProteases are a kind of enzymes which are necessary to preserve homeostasis in cell. So far, 12 protease therapies have been proposed by the U.S. FDA (Food and Drug Administration) . Based oProtease peptide inhibitors that occur in scorpion venoms have broad applications in medicine. Accordingly, proteases and protease inhibitors have important affects in pharmacology .Protease inhibitors (PIs) are kinds of proteins or peptides which can be used to inhibit the catalytic activity of proteolytic enzymes . SPIs haKunitz-type inhibitors are a group of serine protease inhibitors that are specified by a conserved spacing between their cysteine residues. There are one or more Kunitz domains in the inhibitor and these possess a typical disulfide bonding pattern ,176. KunAll the Kunitz-type protease inhibitors of scorpions have been recognized by the primary amino acid sequences of some inhibitor peptides that have been found in the NCBI protein data bank .Ascaris-type peptides usually possess a conserved structure with four short β-strands organized in two approximately vertical β-sheets and stabilized by five disulfide bridges: C1–C7, C2–C6, C3–C5, C4–C10, and C8–C9 .The sequences of all the Ascaris-type protease inhibitors of scorpions which have been found in NCBI protein data bank .V1.3 [V1.3 channel) and it was found that six of seven scorpion toxins, excepting rBmKTT-3, which had weak activity, inhibited ~50–80% of Kv1.3 channel currents at a concentration of 1 μM [Some of Kunitz-type protease inhibitors of scorpions have been found to inhibit potassium channel KV1.3 . Seven K of 1 μM .Hemiscorpius lepturus transcriptome were coded as serine proteinase (KX932440 and KX932441 [The serine proteinase inhibitor (serpin) superfamily participates in various necessary biological processes such as blood coagulation, complement activation, fibrinolysis, angiogenesis, inflammation, and tumor suppression. The members of this superfamily are expressed in a cell-specific manner . BecauseKX932441 .Scorpiops jendeki Ascaris-type protease inhibitor), SjAPI-2 (Scorpiops jendeki Ascaris-type protease inhibitor 2), CtAPI (Chaerilus tricostatus Ascaris-type protease inhibitor), and BmAPI (Buthus martensii Ascaris-type protease inhibitor) [However, four putative serine protease inhibitors were discovered from the venom of three scorpion species including SjAPI (hibitor) .The risk of scorpion stings in rural areas is higher than in urban regions, and also more common in summer . Much atScorpions are the most dangerous venomous animals for humans after snakes (venomous snakes) .Climatic conditions, dryness, and heat are factors that increase the threat of scorpion stings . KhuzestHemiscorpius lepturus, Androctonus crassicauda, Mesobuthus eupeus, Odontobuthus doriae, Hottentotta saulcyi, Hottentotta schach, Compsobuthus matthiesseni, Olivierus caucasicus, Orthochirus scrobiculosus, and Apistobuthus pterygocercus are significant species in terms of medical and pharmacological relevance. Among Iranian scorpions, Androctonus crassicauda and Hemiscorpius lepturus have the highest risk of envenoming humans [Androctonus crassicauda  extracts venoms of i et al. ,48 and Fi et al. , specime Kovařík  and Navi Kovařík ,35, specIn conclusion, studying toxins of scorpion species from Iran is a way to present patterns and connection between species of scorpion and their venoms, which could be useful for understanding the molecular and functional diversities of scorpion venom sources, their evolutions, and probably the connection between scorpion species and their toxins.


YKL-40 level was correlated with the length of hospital stay , the levels of inflammatory markers—that is, blood leukocytes , plasma C-reactive protein , and interleukin-6 , and maximum plasma creatinine level . In conclusion, plasma YKL-40 levels were found to be elevated during acute PUUV infection and correlated with the overall severity of the disease, as well as with the degree of inflammation and the severity of AKI.Most cases of hemorrhagic fever with renal syndrome (HFRS) in Europe are caused by the Puumala hantavirus (PUUV). Typical features of the disease are increased vascular permeability, acute kidney injury (AKI), and thrombocytopenia. YKL-40 is an inflammatory glycoprotein involved in various forms of acute and chronic inflammation. In the present study, we examined plasma YKL-40 levels and the associations of YKL-40 with disease severity in acute PUUV infection. A total of 79 patients treated in Tampere University Hospital during 2005–2014 were studied. Plasma YKL-40 was measured in the acute phase, the recovery phase, and one year after hospitalization. Plasma YKL-40 levels were higher during the acute phase compared to the recovery phase and one year after hospitalization (median YKL-40 142 ng/mL, range 11–3320, vs. 45 ng/mL, range 15–529, vs. 32 ng/mL, range 3–213,  Hantaviruses have caused hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia, and hantavirus cardiopulmonary syndrome (HCPS) in America ,2,3. TheThe typical features of PUUV infection are increased vascular permeability, acute kidney injury (AKI), and thrombocytopenia, with the last one rarely causing serious hemorrhages ,6,7,8,9.A central phenomenon in hantavirus infections is increased capillary permeability, leading to vascular leakage ,8. The eComplement activation, as well as several biomarkers, including plasma interleukin (IL)-6, indoleamine 2,3-dioxygenase, pentraxin-3, soluble urokinase-type plasminogen activator receptor (suPAR), and cell-free DNA have been shown to predict the outcome of PUUV infection ,22,23,24YKL-40, also known as chitinase 3-like protein 1 (CHI3L1), is an inflammatory glycoprotein secreted by a variety of cells, particularly by activated macrophages and neutrophils, in different tissues with inflammation ,32. It iIn this study, we examined the associations of YKL-40, a multifunctional inflammatory factor, with inflammation, AKI, and the overall disease severity in HFRS induced by PUUV.The study cohort originally consisted of 86 consecutive patients with acute, serologically confirmed PUUV infection treated in Tampere University Hospital in Finland during January 2005 to November 2014. Plasma samples for YKL-40 measurements were available from 79 patients, and these patients comprised the final study cohort. A detailed medical history was obtained and careful physical examination was performed during the acute phase of the disease. All patients provided a written informed consent, and the study was approved by the Ethics Committee of Tampere University Hospital .n = 7), asthma/chronic obstructive pulmonary disease (n = 4), gastritis/reflux disease (n = 4), rheumatoid arthritis (n = 3), coronary artery disease (n = 2), type 2 diabetes (n = 2), type 1 diabetes (n = 1), and transient ischemic attack (n = 1). Some of the patients had more than one disease, but none had a known kidney disease or chronic renal insufficiency.The median age of the patients was 41 (range 21–74) years, and 48 patients (61%) were males. Of the patients, 24 (30%) had one or more of the following diseases diagnosed before acute PUUV infection: hypertension  , and/or 6 cells/L (about 3–5 cells by high power field). The dipstick test for glucose detects glucosuria from glucose level 3–5 mmol/L upwards. Glucosuria 3+ corresponds to a urine glucose level higher than 30 mmol/L.Plasma creatinine was measured daily during hospitalization, with a median of five (2–13) measurements per patient, by a Cobas Integra analyzer . A urine dipstick test was performed on admission to hospital. The urine dipstick analyses were performed by automated tests based on refractometry (Siemens Clinitec Atlas or Advantus). The dipstick assay detects albumin, and it does not react with immunoglobulins, immunoglobulin light chains, or tubular proteins. The sensitivity of the assay to urine albumin is 0.15–0.3 g/L (U-Alb 1+), ≥1 g/L (U-Alb 2+), and ≥3 g/L (U-Alb 3+). The assay for hematuria detects heme pseudoperoxidase activity, and therefore, it detects red cell casts and dysmorphic red cells as well. The sensitivity of the assay is about 10 × 10Plasma samples for the measurement of YKL-40, resistin, leptin, and adiponectin concentrations, as well as CRP and IL-6 levels were collected between 7:30–8:30 am, with a median of two (1–5) times during hospitalization. The follow-up samples were obtained at a median of 15 (range 7–21) days after discharge from hospital in 74 patients, and one year after hospitalization in 67 patients. Plasma YKL-40, resistin, leptin, adiponectin, CRP, and IL-6 concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) using reagents from R&D Systems Europe Ltd., Abingdon, UK  and from eBioscience Inc, San Diego, CA, USA (IL-6). The detection limit and interassay coefficient of variation were 15.6 pg/mL and 4.2% for YKL-40, 15.6 pg/mL and 8.5% for resistin, 15.6 pg/mL and 5.3% for leptin, 15.6 pg/mL and 6.0% for adiponectin, 3.9 pg/mL and 5.7% for CRP, and 0.39 pg/mL and 4.8% for IL-6. For adiponectin, the test detects total adiponectin.Blood cell counts were determined by hematological cell counters . Other analytical procedures were carried out with an automated chemistry analyzer using the routine procedure. The highest or lowest values (as appropriate) of the various variables measured during the hospital stay were designated as the maximum or minimum values. All routine laboratory analyses were performed by the Laboratory Centre of Pirkanmaa Hospital District (later named Fimlab Laboratories), Tampere, Finland.2).Here, shock is defined by a fall in systolic blood pressure under 90 mmHg, together with the clinical symptoms of shock. Body mass index (BMI) was calculated as the ratio of weight (kg) to squared height  statistical software .Medians and ranges are given for continuous variables, and numbers and percentages for categorical variables. Spearman’s rank correlations were calculated. Categorical data were analyzed using the The clinical characteristics and laboratory findings of the patients are shown in p < 0.001 for both compared to the acute phase) (p = 0.188).Plasma YKL-40 was elevated during the acute phase, compared to the values in the recovery phase and one year after the infection. e phase) B. The map = 0.004), but not with the levels of the two other adipokines, adiponectin and leptin .We then divided the patients into two groups according to the maximum plasma YKL-40 level: YKL-40 ≤ 142 ng/mL and YKL-40 > 142 ng/mL (142 ng/mL was the median in the study population). The patients with higher YKL-40 levels had a more severe AKI and higher levels of the inflammatory markers , compared to the patients with lower YKL-40 concentrations . The patp = 0.301), or between patients with no hematuria or hematuria level 1+, 2+, or 3+ , or between patients with and without glucosuria .There was no difference in plasma YKL-40 level between patients with no proteinuria or proteinuria level 1+, 2+, or 3+ (YKL-40 median 75, range 26–1296, ng/mL vs 128, range 36–1836, ng/mL vs 178, range 32–2223, ng/mL vs 166, range 12–3321, ng/mL, The present study shows that plasma YKL-40 levels are elevated during acute hantavirus infection caused by PUUV. The concentrations measured during the acute illness were markedly higher than the values measured after hospitalization, which, in turn, corresponded to the YKL-40 levels of local healthy subjects in our previous studies ,37. To oYKL-40 or CHI3L1 is a heparin- and chitin-binding glycoprotein with a molecular weight of 40 kDa, and is secreted by a variety of cells, particularly activated macrophages, neutrophils, and vascular smooth muscle cells on sites of inflammation ,32. It iPreviously, YKL-40 levels have been reported to predict the outcome of some bacterial infections. YKL-40 level was found to be connected with the severity of community acquired pneumonia and the outcome of pneumococcal bacteremia, as well as survival in sepsis ,43,44. FThe maximum plasma YKL-40 level was correlated with the inflammatory markers—that is, blood leukocyte count and plasma CRP and IL-6 levels. This is in accordance with the perception of YKL-40 as an early inflammatory marker. This finding is also in concordance with previous studies, reporting a positive correlation of YKL-40 with IL-6 or CRP in some other inflammatory or infectious conditions ,36,39,46Increased capillary permeability is a central feature in hantavirus infections ,8, but tThe plasma YKL-40 level was associated with the severity of AKI, evaluated by the highest plasma creatinine level. Previously, urine YKL-40 has mainly been studied in AKI. Increased urine YKL-40 has been found to predict AKI or death in hospitalized patients . MarathoIn patients with diabetes, elevated plasma YKL-40 levels are associated with increasing amounts of albuminuria ,32. In tIn conclusion, plasma YKL-40 levels were found to be associated with the severity of Puumala hantavirus infection in this study. It was connected with the overall severity of the disease, the level of inflammation, as well as the severity of AKI.


The relationship for the highly biocompatible Ti-15Zr-4Nb-4Ta alloy was also linear. The mechanical strength and ductility of specimens that were cut from various osteosynthesis devices were excellent and their microstructures consisted of fine structures, which were considered to be related to the excellent durability. These results are expected to be useful for the development of implants suitable for the skeletal structure of patients using three-dimensional (3D) layer manufacturing technologies.To develop orthopedic implants that are optimized for each patient’s needs or skeletal structure (custom-made implants), evaluations of the bending strength, bending stiffness, and durability of various types of conventional osteosynthesis devices have become important. Four-point bending tests and compression bending tests of osteosynthesis devices , short femoral nails, and metaphyseal plates) were carried out to measure their bending stiffness, bending strength, and durability. The bending stiffness of bone plates, intramedullary nails, spinal rods, CHSs, short femoral nails, and metaphyseal plates increased with increasing bending strength. The durability limit of various types of osteosynthesis devices linearly increased with increasing bending strength. The relationship (durability limit at 10 The demand for osteosynthesis devices used for the fixation of fractured bones has been increasing yearly, along with the increase in the number of elderly patients with bone fractures, such as osteoporotic fracture. Regarding the evaluation of the mechanical performance of osteosynthesis devices, Arbeitsgemeinschaft für Osteosynthesefragen (AO) in Switzerland states that sufficient strength and stiffness are required to withstand the load that is applied during clinical bone reduction processes. As the metallic materials for osteosynthesis devices, stainless steel, cobalt (Co)-chromium (Cr)-molybdenum (Mo) alloys, commercially pure titanium (C.P. Ti), and Ti alloys, which provide good balance between strength and stiffness, as well as excellent fatigue properties, have been used. The corrosion resistance and biocompatibility of stainless steel (stainless) can be improved by increasing the amount of each of the elements added to the steel, such as Cr and Mo. In addition, fatigue strength can be increased to a level that is equivalent to that of Ti alloys by adding nitrogen (N) and 20% cold working. The fatigue strength of an industrial Ti material can be increased by increasing the amounts of trace elements, such as oxygen (O) and iron (Fe), whereas the fatigue strength of C.P. Ti grade 4 (C.P. Ti G 4) can be made similar to that of Ti alloys by 20% cold working . Ti alloThe development of orthopedic implants that are optimized for each patient’s needs or skeletal structure (custom-made orthopedic implants) has been made possible, owing to advances in fabrication techniques ,6,7,8. AThe breakage of metallic osteosynthesis devices in clinical use, namely, bone plates ,12,13, iIn this study, we focused on the development of test jigs and the optimization of test conditions for mechanical tests while using various osteosynthesis devices to obtain mechanical data in accordance with the above international and national standards. Specifically, we examined the relationships between the bending strength and bending stiffness and between the bending strength and the durability of various osteosynthesis devices to clarify the effects of base materials on their properties, such as the relationships between tensile strength and Young’s modulus and between the tensile strength and fatigue strength obtained by room-temperature tensile and fatigue tests of raw materials. One of the purposes of this study is to develop a method of estimating the durability from strength by obtaining relational equations for various osteosynthesis devices. Another purpose is to promote the development of implants that are suitable for the skeletal structures of patients using 3D layer manufacturing technologies. Therefore, we examined the bending strength, bending stiffness, and durability of various types of conventional osteosynthesis devices used as parts that are subjected to loading. Four-point bending tests and compression bending tests of osteosynthesis devices  were carried out to measure the bending stiffness, bending strength, and durability, in accordance with JIS T 0312 and JIS T 0313. In addition, the room-temperature mechanical properties of miniature specimens cut from each osteosynthesis device and their microstructure were examined. The results that were obtained in this study will be useful for developing highly durable devices using new technologies, such as 3D printing.The following osteosynthesis devices used worldwide were examined in this work. Regarding the number of holes in the bone plates, eight holes were mainly used in accordance with ISO 9585, ASTM F382, and JIS T 0312. The osteosynthesis devices that were made of Ti-15Zr-4Nb-4Ta (Ti-Zr) alloy, which are described below, were machined to the same shape as the Ti-6Al-4V (Ti-6-4) alloy devices for comparison among the different Ti alloys. (1) Straight plates  manufactured by (i) DePuy Synthes {C.P. Ti G 4; broad and narrow LC-DCPs , condylar plate , broad, narrow, and small LC-LCPs , LCP reconstructions , 1/3 circular small (441-380), and mini-DCP (443-580); lengths, 142, 142, 156, 152, 144, 103, 114, 151, 100, 42; widths, 17.5, 13.5, 16, 17.5, 13.5, 11, 10, 12, 9, 5; thicknesses, 6, 4.6, 5.6, 5.2, 4.6, 3.3, 3.1, 3, 1, 1.5 mm, respectively, and stainless; condylar plate (237-960), 1/3 circular small (241–380), mini-DCP (243–580)}; (ii) Zimmer Biomet ; (iii) MDM ; (iv) Stryker ; (v) Mizuho ; (vi) Teijin Nakashima Medical . (2) Intramedullary femoral nail rods manufactured by (i) DePuy Synthes ; (ii) Zimmer Biomet ; (iii) Stryker ; (iv) Smith & Nephew .(3) Spinal rods manufactured by (i) Medtronic ; (ii) Zimmer Biomet ; (iii) Robert Reid ]. (4) CHSs manufactured by (i) DePuy Synthes ; (ii) Zimmer Biomet ; (iii) MDM ; (iv) Stryker ; (v) Teijin Nakashima Medical ; (vi) Mizuho ; (vii) Japan Medicalnext . (5) Short femoral nails manufactured by (i) Stryker ; (ii) Zimmer Biomet ; (iii) Smith & Nephew ; (iv) DePuy Synthes .(6) Epiphyseal plates manufactured by (i) Stryker ; (ii) DePuy Synthes .Three each of the bone plates, intramedullary nail rods, spinal rods, CHSs, short femoral nails, and metaphyseal plates were prepared for each bending test. At least six osteosynthesis devices were prepared for the durability test.An Instron 8874 mechanical testing system was used for each bending test. Four-point bending tests and compression bending tests were conducted on various osteosynthesis devices, in accordance with JIS T 0312 and JIS T 0313, respectively. In particular, we manufactured jigs that fit the osteosynthesis devices with various shapes that were used in this work. The test conditions were controlled using a personal computer. The bending stiffness and bending strength were calculated using Instron mechanical testing software (Bluehill 2). Details of the bending tests of the various osteosynthesis devices are given below. The mean and standard deviation were calculated from the results of bending tests of three specimens of each type of osteosynthesis device.2) was calculated as E = (2h + 3k) × A × h2/12, where A is the maximum slope of the linear elastic portion of the L–D curve, h is the distance between the nearest supporting roller and the loading roller (m), and k is the distance between two loading rollers (m). For the four-point bending tests of intramedullary nail rods, the central region of the rod was cut so that the length of the rod was ~130 mm. The four-point bending test of the thus obtained specimen was carried out with the ratio (distance between loading rollers):(distance between supporting rollers) = 1:3 (38:114 mm). For spinal rods, the rod was cut to a length of ~100 mm and the h:k ratio was 30:60 (mm), since the spinal rods had a small diameter. The compression bending strength (M) and bending structural stiffness (E) for intramedullary nail rods and spinal rods were measured using the same calculation formulas, as above.The bending stiffness, bending strength, and durability of bone plates, intramedullary nail rods, and spinal rods were measured by the four-point bending test. Five Instron 8874 mechanical testing systems were used for each durability test. The test conditions were controlled using the Instron software (Max), and changes in the waveform during the test were constantly monitored. M–N curves  were measured for the four-point bending durability test and the compression bending durability test. The maximum bending moments (M) were calculated as M = h × Pm/2 for the four-point bending durability test and M = L × Pm for the compression bending durability test, where Pm is the maximum load (N) in the four-point bending durability test or compression bending durability test. 6 cycles, was determined.Using the four-point bending test jigs, the durability of bone plates, intramedullary nail rods, and spinal rods was evaluated under the following conditions: (distance between loading rollers):(distance between supporting rollers) ratio = 1:3, except for spinal rods, h:k ratio = 1:2 for spinal rods, sinusoidal waves, and load ratio R (minimum load/maximum load) = 0.1. For bone plates, sinusoidal waves with a frequency in the range of 1–8 Hz (mainly 5 Hz) were applied to examine the effect of the frequency. For intramedullary nail rods and spinal rods, sinusoidal waves with a frequency of 3 Hz were used. From the obtained M–N curves, the durability limit, which is the maximum moment corresponding to 1 × 106 cycles. From the obtained M–N curve, the durability limit after 1 × 106 cycles was determined by the same method. A compression bending durability test was carried out using similar jigs to evaluate the static compression. Sinusoidal waves with frequencies of 3 or 5 Hz (CHSs), 3 Hz , and 1 Hz  were applied at a stress ratio R = 0.1 for more than 1 × 10Three uniform rod specimens  were cut from each bone plate, intramedullary nail rod, spinal rod, CHS, short femoral nail, and metaphyseal plate, in accordance with the shape of the specimen. The strain rate was set to 0.5%/min until failure. We analyzed the cross-sectional microstructure by optical microscopy and transmission electron microscopy (TEM). Nitric hydrofluoric acid solution (3%) was used as an etching agent. The 3-mm-diameter disc specimens were electropolished with 95% methanol +5% perchloric acid solution.We calculated the correlation coefficients (r) between the bending stiffness and the bending strength and between the durability limit and the bending strength for each bone plate, intramedullary nail rod, spinal rod, CHS, short femoral nail, and metaphyseal plate. Additionally, linear regression analysis was performed between the bending stiffness and the bending strength, and between the durability limit and the bending strength, in both cases assuming a line passing through the origin. Moreover, the M–N curve, the durability limit of various osteosynthesis devices, and its standard deviation were calculated with statistical analysis software that is based on JSMS-SD-06-08 .2 = 0.99). From these results, it was clarified that the h:k ratio affects the four-point bending structural stiffness and bending strength, but in many tests, h:k = 1:1 recommended by ASTM F382 and JIS T 0312 is employed. 2 = 0.73–0.92), which were measured under a condition of h:k = 1:1. Regarding the relationship between the four-point bending structural stiffness and the bending strength, we obtained [bending structural stiffness (N·m2)] = 0.32 × [four-point bending strength (N·m)] (R2 = 0.88) by regression. The relationship for the highly biocompatible Ti-Zr alloy was also linear. The four-point bending strength is expressed asBecause the recommended h:k ratios in ISO 9585, ASTM F382, and JIS T 0312 are different, we examined the effect of the h: k ratio. σ is the surface stress that is generated by four-point bending, α is the stress concentration factor, and Z is the section modulus of the osteosynthesis device, which depends on the cross-sectional shape and affects the bending structural stiffness and bending strength  for bone plates, spinal rods, and intramedullary nail rods were approximately 0.3  = 0.67 × (bending strength) (N·m) (R2 = 0.85) was obtained by regression. This slope of 0.67 was close to the ratio of the fatigue strength to the tensile strength of the raw metals. The relationship for the highly biocompatible Ti-15Zr-4Nb-4Ta alloy was also linear.The durability limit of various types of osteosynthesis devices linearly increased with increasing bending strength (r = 0.93). The relationship (durability limit at 104. The mechanical strength and ductility of specimens that were cut from various osteosynthesis devices were excellent and their microstructure consisted of fine structures, which are considered to be related to the excellent durability.


Ailuropoda melanoleuca) express transitional characteristics in that they consume bamboos, despite their carnivore-like digestive tracts. Their genome contains no cellulolytic enzymes; therefore, understanding the development of the giant panda gut microbiome, especially in early life, is important for decoding the rules underlying gut microbial formation, inheritance and dietary transitions. With deep metagenomic sequencing, we investigated the gut microbiomes of two newborn giant panda brothers and their parents living in Macao, China, from 2016 to 2017. Both giant panda cubs exhibited progressive increases in gut microbial richness during growth, particularly from the 6th month after birth. Enterobacteriaceae dominated the gut microbial compositions in both adult giant pandas and cubs. A total of 583 co-abundance genes (CAGs) and about 79 metagenomic species (MGS) from bacteria or viruses displayed significant changes with age. Seven genera  showed great importance with respect to gut microbial structural determination in the nursing stage of giant panda cubs. Furthermore, 10 orthologous gene functions and 44 pathways showed significant changes with age. Of the significant pathways, 16 from Escherichia, Klebsiella, Propionibacterium, Lactobacillus, and Lactococcus displayed marked differences between parents and their cubs at birth, while 29 pathways from Escherichia, Campylobacter and Lactobacillus exhibited significant increase in cubs from 6 to 9 months of age. In addition, oxidoreductases, transferases, and hydrolases dominated the significantly changed gut microbial enzymes during the growth of giant panda cubs, while few of them were involved in cellulose degradation. The findings indicated diet-stimulated gut microbiome transitions and the important role of Enterobacteriaceae in the guts of giant panda in early life.Adult giant pandas ( Escherichia, Shigella, and Streptococcus  in its gut as an adult, mostly from Firmicutes or Proteobacteria, and dominated by tococcus . Howeverteroides , are conBacillus  and Close bamboo , are wide bamboo . Furthere bamboo . This vae bamboo . In conte bamboo  or surfae bamboo  secretedMicrobacterium, Clostridium, Citrobacter, Delftia, Enterobacter, Klebsiella, Raoultella, Serratia, Enterococcus, Lactobacillus, and Staphylococcus) showed significant differences between the S1 and S3/S4 groups and among S1, S2, S3, and S4, respectively, none of which belong to potentially cellulolytic species. Furthermore, abundant genes related to hemicellulose degradation were detected  and parents , to demonstrate their gut microbiome transitions in the 1st year after birth.Presently, a giant panda family with four members is living in Macao Giant Panda Pavilion . The two cubs were born 3 h apart. They were fed with breast milk until 1.5 months after birth, and then fed mainly with formula milk powder (mixtures of dog and cat milk powders). Due to the specificity of species and sampling times for the study, we obtained a total of 22 fecal samples from the two giant panda cubs in the 1st year of the nursing stage, and we also collected 11 fecal samples from the parents after the cubs were born.After fecal samples were collected, we immediately extracted microbial DNA; all experimental steps were performed in a biosafety cabinet where possible. Owing to the huge differences in fecal size and components between cub feces and adult feces, we adopted different strategies to extract the microbial DNA. Approximately 1.0 g cub fecal samples were weighed and subjected to microbial DNA extraction, as described in the manual of the Power water DNA isolation kit . Approximately 20 g adult giant panda samples were weighed into a sterilized BagFilter partitioned by a 280 μm pore size membrane. Then, we homogenized the BagFilter for approximately 3 min in InterScience BagMixer 400SW . Following that, the filtrates were guided into a sterilized rapid flow Biofilter with a 0.22 μm pore size filter membrane to collect bacteria, fungi and other organisms or materials with a size larger than 0.22 μm. Via the two-level cascaded filtrations, we enriched fecal microbes on the 0.22 μm pore size membrane, and then extracted microbial DNAs on the membrane using the Power water DNA isolation kit for subsequent metagenomic sequencing. We added the sample collection time to individually assigned codes to yield DNA sample names.Before sequencing, the extracted microbial DNAs were fragmented to 250 ~ 500 base pairs (bps) using Covaris E220 and AMPure XP beads (AGENCOURT). Next, the DNA fragments were repaired to generate a blunt end and we further modified its 3′end to obtain a dATP sticky end. Both ends of the DNA fragments were ligated with dTTP tailed adapter sequences and we then amplified these clones for eight PCR cycles. A single-strand circularization process was subsequently performed to generate a single-strand circular DNA library. Afterward, libraries were sequenced on the BGISEQ-500 platform in a paired-end model to obtain a read 100 bp in length.e-value ≤ 1e-5. Here, when the gene length coverage mapped by the Nt database was less than 50%, the gene was assigned “Unclassified” status. For each sample, taxonomic sources of genes were annotated by aligning high quality microbial reads to the non-redundant gene set using Bowtie2 (v2.2.5)  , to remo(v2.2.5) , and gen(v2.2.5) . Species(v2.2.5) . Microbe(v2.2.5)  was used(v2.2.5)  was used(v2.2.5)  and “ape(v2.2.5) , respectWe used Pearson’s correlation coefficients (PCCs) to define the distance from a randomly picked ‘seed’ gene and other genes correlated with the seed gene; such genes were considered as the “canopy” . Canopiet-test, p > 0.05). Finally, the significant spearman correlation network of microbial genera was displayed in Cytoscape (v3.5.1) (n = 500 permutations). The out-of-bag (OOB) error rate was used to assess model accuracy. Furthermore, microbial importance was measured by the mean decrease in the Gini index at each node of the constructed model, where a higher value for the microbe means that it is more important in determining the gut microbial structure of giant panda.We used two approaches to discover structurally important microbes. The first approach focused on microbial genera with more than 70% occurrence frequency in all investigated samples. Correlations between the ‘stable’ genera were measured by their abundance profiles in samples. We then removed non-significant correlations ((v3.5.1) . Secondlp < 0.05, by Holm method (The non-redundant gene set was annotated by blasting translated proteins of the non-redundant genes against the Orthologous Groups of proteins (COG) database (v20090331)  and Kyotm method ].p < 0.05, by Holm method). The network of significant ECs was displayed in Cytoscape (We obtained enzyme commission numbers (ECs) of genes by KEGG annotation. The abundance of ECs was determined by the sum of assigned gene abundances, and significantly changed ECs between age groups were then detected by Wilcoxon test : gut microbial evenness and diversity increased as the cubs developed, yet reverted back to the level of their parents after 6 months of age , Leclercia, Pantoea, Campylobacter and Fusobacterium, and several genera in viral families Podoviridae (P22virus), Siphoviridae (Lambdavirus), Myoviridae  were also in high abundance from birth or enriched soon after birth and stayed in high abundance in parental samples. As a complement to reported prime eukaryotic viral families, for instance Genomoviridae, Picornaviridae, Picornavirales, and Picobirnavirus-like, in the gut of adult giant panda  pathogens, such as urfactin , both Lativities , and aid lactose . During R2 = 0.484, p = 0.001)  Figure . Like th) Figure , and grap < 0.05; LDA score > 2.0) . Pantoea ananatis, which shows quorum sensing and drives gene expression, was significantly enriched in C2 groups, and bacteriophages linked with Lactobacillales  were also significantly increased in the C2 group, yet they dramatically decreased in parental groups.It was difficult to distinguish representative gut microorganisms between 0 ~ 1.5-month-old (C0) and 1.5 ~ 6-month-old (C1) cubs. Nevertheless, when comparing the gut microbial compositions between C0/C1 cubs and 6 ~ 9-month-old cubs C2) (with LDA score > 4.0) at the microbial family level, we discovered that Enterobacteriaceae is the representative family of C0/C1 cubs, and Lactobacillaceae and Campylobacteraceae are the representative families of C2 cubs. Remarkably, the representative gut microbiota families of C2 cubs are consistent when compared with either C0/C1 or F/M. In addition, compared with the gut microbiota of C2 group cubs, the representative families of paternal and maternal gut microbiota are Enterobacteriaceae and Clostridiaceae, respectively  and most of them occurred at low abundance . As shown in Figure Klebsiella, Edwardsiella, Serratia, and Raoultella), belonging to the order Enterobacterales, were negatively correlated with many other stable genera, and their abundances gradually decreased during the growth of giant panda cubs. Notably, 18 genera  between age groups  and 357,909 (34.39%) non-redundant genes were annotated in COG database and KEGG database, respectively. Only 10 orthologous functions showed significant changes  between age groups  , we detected extensive increases of abundance of KOs relating to starch degradation from 2 months after birth, particularly at 6 ~ 9 months of age Figure . DespiteAlthough increasing numbers of studies of the significant gut microbes or pathways in giant panda have been reported in recent years , few focErwinia, Providencia, Edwardsiella, Pectobacterium, and Serratia. Worthy of mention is Pectobacterium, a group of bacteria that produce pectolytic enzymes to hydrolyze pectins of bamboos. Meanwhile, the reported abundant genera Leuconostoc and Stenotrophomonas ; Campylobacter significantly enriched ‘purine metabolism and pyrimidine metabolism,’ ‘carbon metabolism,’ ‘RNA polymerase,’ and the NOD-like receptor signaling pathway in 6 ~ 9-month-old cubs, which facilitated cell cycle and strengthened the environmental interplays of gut microbiota. Here, I stress that Lactobacillus and Campylobacter also enriched in gut microbiota of 1.5 ~ 6-month-old cubs, so they were excluded from the list of microbes that could discriminate the gut microbiota architecture among 0 ~ 1.5-month-old cubs (C0), 1.5 ~ 6-month-old cubs (C1), 6 ~ 9-month-old cubs (C2) and parents. However, Campylobacteraceae was reported to be scarce in >4-month-old captive pandas , rarely reported in previous studies  was the only infectious pathway that significantly changed with age. Taken together with the enrichment of (opportunistic) pathogens and infectious pathways and large decreases of anti-pathogenic Bifidobacterium and Lactobacillus  was enriched, after 12 months old of age , which led to a fast increase of gut microbial richness and abundance Figure . Despited of age , the biod of age . This suP22virus, Lambdavirus, P1virus, P2virus, and T4virus in high abundance in cubs and adult giant pandas, whereas some abundant genera, namely Epsilon15 virus, Nona33 virus, Rtp virus, G7c virus, and Tl2011 virus .All raw reads of samples are available in National Center for Biotechnology Information (NCBI) database, and have been deposited under BioProject ID: The sample collection and all the experiments were performed in a manner to minimize risk to the giant pandas and the environment. All experimental protocols of this study were approved by Institute of Chinese Medical Sciences - Animal Ethics Committee (ICMS-AEC) of the University of Macau.MG and SL designed the research. MG and JwC performed the research. MG, JwC, JM, YW, and GF contributed to analytic tools. MG, JC, YF, QL, LP, and LZ analyzed data. MG and SL wrote the paper. All authors read and approved the final manuscript.LZ and JC were employed by company Realbio Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We have developed a silver-copper oxide heteronanostructure able to combine the plasmonic-enhanced properties of Ag nanostructures with the visible-light driven photoresponse of CuO nanoarchitectures. The entangled Ag-CuO heteronanostructure exhibits a broad absorption towards the visible-near infrared (NIR) range and achieves total photo-oxidation of n-hexane under irradiation with different light-emitting diodes (LEDs) specific wavelengths at temperatures below 180 °C and outperforming its thermal catalytic response or its silver-free CuO illuminated counterpart.Volatile organic compounds (VOCs) are recognized as hazardous contributors to air pollution, precursors of multiple secondary byproducts, troposphere aerosols, and recognized contributors to respiratory and cancer-related issues in highly populated areas. Moreover, VOCs present in indoor environments represent a challenging issue that need to be addressed due to its increasing presence in nowadays society. Catalytic oxidation by noble metals represents the most effective but costly solution. The use of photocatalytic oxidation has become one of the most explored alternatives given the green and sustainable advantages of using solar light or low-consumption light emitting devices. Herein, we have tried to address the shortcomings of the most studied photocatalytic systems based on titania (TiO Among these contaminants, the exposure to volatile organic compounds (hereafter VOCs) is recognized as a serious hazard to human health contributing to skin, respiratory, and cancer diseases ,2,3. Eveeratures ,37,38,39eratures ,43,44,45 of VOCs ,49,50,51 Mn, Co) ,56,57,582) and/or reducing the electron-hole recombination rates of metal transition oxides  [2 hybrid systems [To overcome these drawbacks, current research interests in VOCs remediation are focused on the development of hybrid nanomaterials combining metal oxides, metal transition oxides and/or noble metals with photocatalytic response expanded towards the visible-NIR ranges ,67,68,69 Co, Cu) ,88,89,90 Co, Cu) ,94,95,96 Co, Cu) ,88,89,90 Co, Cu) ,103,104. systems ,99,100.xOy systems [2 [In the present work, we aimed at exploring the synthesis of a hybrid heterostructured catalyst combining the plasmonic properties of Ag and the p-type semiconductor capabilities of CuO ,103,104. systems ,106,107  systems ,111,112, systems ,114,115 stems [2 ,108 havestems [2 ,114,116,stems [2 ,119,120.3)2·3H2O  and AgNO3  were dissolved in 3 mL of deionized water. The resulting silver-copper solution was added to an aqueous solution of NaOH 3 M  with vigorous stirring for 6 h under an inert Ar atmosphere. The resulting black adduct that was vacuum filtered, washed with water, and dried at 100 °C for 2 h. The solid was calcined at 350 °C for 6 h. In order to obtain silver-free CuO nanostructures, an analogous synthesis protocol was followed but skipping the addition of the silver salt precursor. The synthesis of the photocatalysts has been performed at the platform of Production of Biomaterials and Nanoparticles of the NANBIOSIS ICTS, Spain, more specifically by the Nanoparticle Synthesis Unit of the CIBER in BioEngineering, Biomaterials & Nanomedicine (CIBER-BBN).The Ag-CuO heteronanostructures were synthesized following a protocol reported elsewhere . Cu(NO3)3 electron microscope operated at 200 kV. Elemental analysis was carried out with Energy Dispersive Spectroscopy (EDS)  detector using single point and scanning profiles. The samples were drop-casted onto Ni mesh grids. The N2 adsorption/desorption analyses were performed with the aid of a Micromeritics ASAP2020 analyzer . 80–100 mg of the catalyst was degassed at 90 °C for 12 h. The surface area was determined using the Brunauer–Emmett–Teller (BET) method rendering a value of 4.7 m2·g−1 for the Ag-CuO tubular heterostructures. Scanning electron microscopy (SEM) analysis was carried out with FEI-Inspect S50 equipment . X-ray diffraction patterns were obtained in a PANalytical Empyrean equipment  in Bragg Brentano configuration using Cu-K radiation and equipped with a PIXcel1D detector. The absorption spectra were acquired with a JASCO V-670 UV-VIS-NIR spectrophotometer  and the aid of an integrated sphere accessory.Transmission electron microscopy (TEM) analysis was carried out using a T20–FEI microscope . Aberration corrected scanning transmission electron microscopy (STEM) images were acquired using a high angle annular dark field detector in a FEI XFEG TITAN microscope  at 300 kV equipped with a CETCOR Cs-probe corrector. High-resolution transmission electron microscopy (HR-TEM) images were acquired with the aid of a FEI TITAN3 (height × width × length). The cell was illuminated by two high power LEDs (LedEngin) cooled with the aid of custom-designed fans. Different LEDs wavelengths were individually tested. Each LED operated with different power: 405 nm (3.9 W), 460 nm (2.2 W), and 940 nm (3.2 W). Selected light irradiances ranged from 7 to 11,000 mW/cm2  and LED powers were programmed with the aid of an external power supply unit . The temperature of the catalytic bed containing 300 mg of Ag-CuO during irradiation was monitored with the aid of a K-type thermocouple. The photocatalytic experiments were performed with a total flow of 50 mL·min−1 of gas containing 200 ppm of n-hexane . Conventional heating experiments were carried out with a home-built system consisting of an aluminium holder designed to heat the same reactor area as in the quartz reactor used for the photocatalytic test [2, O2, and synthetic air , in order to get the different total flow rates assessed. After an equilibration period of 30 min that served us to evaluate the adsorption of n-hexane in the dark, the LED lights were turned on for different time intervals and the gas effluent outlet analyzed by gas chromatography . An OV-1 and a PPQ column in line with a thermal conductivity detector (TCD) were employed to separate and detect the different gas compounds. The steady state final concentration achieved was always ≤10 ppm of n-hexane when maximum LED power was used. This steady state was always achieved within minutes regardless of the experimental settings. Under the conditions used, the n-hexane detection limit was 3 ppm and CO2 was the only oxidation product detected. Maximum error in the mass balance closures for carbon and oxygen in this work was ±2%.The experimental setup designed to carry out the photocatalytic degradation of n-hexane has been previously described elsewhere ,122. BriThe morphological evaluation of the Ag-CuO heterostructures by SEM revealed the presence of tubular-shaped structures a 121,12,124. A mIt is also worth mentioning that Ag was identified with different morphologies, including small segregated nanoparticles, rod-shaped anisotropic structures g, and nophoton = K (Ephoton− Eg)1/2 . The estimated band gap energy was calculated at 1.33 eV for the CuO nanostructures and 0.71 eV for the Ag-CuO hybrids  and a monoclinic (C2/c) system, respectively a. The op hybrids d. These 50 (Temperature of reaction required to reach 50% of conversion) remained below 100 °C. These results contrast with the photocatalytic behavior identified for the silver-free CuO counterpart under similar LED irradiation conditions. In this latter case, temperatures above 200 °C were necessary to reach match T50 and a complete n-hexane oxidation was not achieved  required at 405, 460, and 940 nm, respectively. Upon comparison of the three LEDs, it became clear that the photocatalytic efficiency was higher at 460 nm. The irradiation under the LED emitting at 405 nm required almost double irradiance to reach full photo-conversion of n-hexane. We observed a stable photo-response after multiple cumulative reaction runs performed under different LED wavelengths and no evidences of deactivation.Additional photocatalytic experiments were carried out with the Ag-CuO heterostructure under illumination with different LED wavelengths at 460 and 940 nm, respectively. The use of LEDs emitting in the visible and NIR ranges rendered equivalent n-hexane photo-oxidation levels and analogous overlapping light-off curves to the one displayed in xCuyOz that evolves into the corresponding silver and copper oxides counterparts [2O into metallic Ag and oxygen at temperatures in the range of 195–205 °C and enabling partial mobility of silver throughout the CuO tubular scaffold  that remain embedded within the CuO tubular matrix e,g,i can2) is the most accepted plasmon-driven charge transfer mechanism, there exist recent interesting studies claiming the importance of hot holes in other plasmonic-based systems [x, Au-pGaN [Surface plasmons can alternatively decay via non-radiative pathways involving the generation of electron-hole pairs by interband and/or intraband excitations ,81,92,95 systems ,136,137  Au-pGaN , Au nano Au-pGaN , Au nano Au-pGaN ,139,140, Au-pGaN ,137.We tentatively propose a combination of the different photocatalytic mechanisms given the diversity of Ag domains. Indeed, the better photoresponse of the Ag-CuO hybrid in comparison with the CuO nanostructures confirms the important role of silver as plasmonic structure to harvest light in the whole visible to NIR range. Likewise, the close contact between both metal and semiconductor phases has enabled a suitable interfacial contact to promote electron and holes mobilities and minimize undesired recombination and relaxation pathways. In summary, we can conclude that our Ag-CuO represents a very attractive metal/p-type semiconductor candidate with full-spectrum response that can be envisioned as an affordable alternative for green and sustainable photo-assisted chemistry, with special attention to energy and remediation processes.


Generation of human induced pluripotent stem cells (hiPSCs) and their differentiation into a variety of cells and organoids have allowed setting up versatile, non-invasive, ethically sustainable, and patient-specific models for the investigation of the mechanisms of human diseases, including viral infections and host–pathogen interactions. In this study, we investigated and compared the infectivity and replication kinetics in hiPSCs, hiPSC-derived neural stem cells (NSCs) and undifferentiated neurons, and the effect of viral infection on host innate antiviral responses of representative flaviviruses associated with diverse neurological diseases, i.e., Zika virus (ZIKV), West Nile virus (WNV), and dengue virus (DENV). In addition, we exploited hiPSCs to model ZIKV infection in the embryo and during neurogenesis. The results of this study confirmed the tropism of ZIKV for NSCs, but showed that WNV replicated in these cells with much higher efficiency than ZIKV and DENV, inducing massive cell death. Although with lower efficiency, all flaviviruses could also infect pluripotent stem cells and neurons, inducing similar patterns of antiviral innate immune response gene expression. While showing the usefulness of hiPSC-based infection models, these findings suggest that additional virus-specific mechanisms, beyond neural tropism, are responsible for the peculiarities of disease phenotype in humans. Flavivirus genus of the family Flaviviridae includes several arthropod-borne human pathogens that may cause a spectrum of clinical syndromes ranging from a mild febrile illness to severe hemorrhagic fever or neuroinvasive disease. Neurotropic flaviviruses, such as West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV), are maintained in nature in transmission cycles involving mosquito (mainly Culex spp.) or tick (mainly Ixodes spp.) vectors and mammalian or bird amplification hosts, with humans serving as dead-end incidental hosts. Most human infections are asymptomatic or characterized by flu-like illness, while a severe and potentially fatal neuroinvasive disease occurs in a minority of cases, mostly immunocompromised and elderly individuals [The ividuals .Ae. aegypti and other Aedes species mosquitoes, with humans serving as amplifying hosts. Unique among flaviviruses, ZIKV can persist in immune-privileged sites like the testis and be sexually transmitted [Zika virus (ZIKV) has recently drawn attention due to the large outbreaks that occurred in Pacific Ocean countries and the Americas. Like other flaviviruses, ZIKV generally causes asymptomatic infections or a mild febrile illness . Infectinsmitted ,5.In addition to these well-known neurotropic flaviviruses, there has been an increasing number of reported cases of neurological complications caused by viruses, such as dengue virus (DENV) and yellow fever virus (YFV), which usually are associated with hemorrhagic disease ,7.One of the central questions in the search for therapeutic targets for the treatment of flaviviral neurological syndromes is whether these diseases are induced by virus-mediated cytolysis, immune-pathological responses to infection, or a combination of both ,9. AnimaIn this study, we investigated the infectivity, tropism, and replication kinetics of ZIKV, in comparison with WNV and DENV in hiPSCs, hiPSCs-derived neural stem cells (NSCs) and undifferentiated neurons, and the effect of viral infection on host innate antiviral responses. In addition, we exploited hiPSCs to model ZIKV infection in the embryo and during stem cell differentiation towards the neuronal lineage. The results of this study confirmed the tropism of ZIKV for NSCs, but showed that WNV replicated in these cells with much higher efficiency, inducing massive cell death. Although with lower efficiency, all flaviviruses could also infect pluripotent stem cells and neurons, inducing similar patterns of antiviral innate immune response gene expression. While showing the usefulness of hiPSC-based infection models, these findings suggest that additional virus-specific mechanisms, beyond neural tropism, are responsible for the peculiarities of disease phenotype in humans.The different cellular tropism and ability to counteract host cell intrinsic immunity is conceivably one of the key factors that explains the diverse clinical presentation of infections caused by genetically related viruses. This is the case of neurological disease associated with flavivirus infection. In this part of the study, we evaluated and compared the infection and replication efficiency of flaviviruses in pluripotent stem cells and cells differentiated towards the neural lineage. Among flaviviruses, we chose WNV as representative of neurotropic viruses, DENV as representative of viruses associated with hemorrhagic fever, which may cause neurological complications in patients with severe shock syndrome, and ZIKV, which is unique among flaviviruses for its ability to infect the fetus during pregnancy and cause severe developmental defects of the central nervous system. For infection modeling, we used human iPSCs (hiPSCs), which share pluripotency features of human embryonic stem cells, NSCs and immature neurons, which represent the neural progenitor cells and neurons in the developing fetal brain, respectively. hiPSCs clones were generated by reprogramming of erythroblasts expanded from peripheral blood mononuclear cells (PBMCs) with Sendai virus vectors carrying the four Yamanaka factors . Human NSCs and immature neurons were obtained from the neural differentiation of human iPSC clones .NSCs were infected with viruses at the multiplicity of infection (MOI) of 1. MOI was defined as the ratio of the number of infectious virus particles measured as plaque forming units, PFU, or tissue culture infectious dose 50%, TCID50, to the number of target cells that were present in a well. Target cell infection was assessed at 72 h post infection (hpi) by immunofluorescence staining with a monoclonal antibody targeting the flavivirus envelope (E) glycoprotein. The percentage of infected cells was estimated by fluorescence microscopy at 40× magnification by counting the number of positive cells to the total number of cells in five fields of view in triplicate experiments. The average infection rate was estimated as 25% ± 8%, 80% ± 12%, and 15% ± 5% of NSCs infected with ZIKV, WNV, and DENV-2, respectively. Representative confocal microscopy images of ZIKV, WNV, and DENV-2-infected NSCs are shown in Infection efficiency was also evaluated in hiPSCs and in hiPSC-derived neurons . In bothViral replication kinetics was evaluated in time-course experiments of infection at different MOIs in hiPSCs, NSCs, and neurons . Viral RZIKV exerted a cytopathic effect (CPE) not only in NSCs, but also in hiPSCs and neurons infected at different MOIs. An aggressive CPE was observed also in WNV-infected cells, while no CPE was apparent in DENV-2-infected cells at 96 hpi, in agreement with the lower replication efficiency of this virus. Accordingly, measurement of cell viability by the MTT assay showed massive death of NSCs at 96 hpi with WNV, while no significant decrease in cell viability occurred following infection with ZIKV and DENV-2 a. A signTLR3, DDX58/RIG-I, IFIH1/MDA5, and by ssRNA (TLR7); genes encoding interferon (IFN) regulatory factors 3 (IRF3) and 7 (IRF7); interferon type I IFNα (IFNA1) and IFNβ (IFNB1); IFN-stimulated genes with antiviral activity, i.e., IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) and 2 (IFIT2) and Viperin (RSAD2); the mediator of the inflammatory response IL-1β (IL1B). Intrinsic expression of innate antiviral immune response genes, analyzed in cells harvested at different time points after seeding, showed no significant differences, while some variability in mRNA expression levels was observed according to cell differentiation status  activated by viral dsRNA, i.e., n status a.IFIT2 mRNA, which was significantly higher in infected NSCs than in mock-infected control NSCs, induction of expression of most genes was significant at 96 hpi , induced in the highest levels of innate immune response gene transcripts, especially in NSCs, with a particularly strong stimulation of IFNB1 and the effector IFIT2, a critical mediator involved in antiviral immunity.Modulation of genes involved in innate antiviral immune response was analyzed by qRT-PCR on hiPSCs, NSCs, and neurons after infection with ZIKV (MOI 1), WNV (MOI 0.01), and DENV-2 (MOI 1) at 48 and 96 hpi. While no relevant changes in mRNA levels were apparent at 48 hpi, except t 96 hpi b. In NSCIn order to model ZIKV infection during neurogenesis, hiPSCs and NSCs were infected with ZIKV at MOI 0.1 and 1, followed by triggering the differentiation process into NSCs and neurons, respectively. Supernatants were harvested every 24 h until 7 days pi for viral titration by qRT-PCR and TCID50 assay. In hiPSCs infected prior to their differentiation into NSCs, ZIKV replicated efficiently and induced massive cell death. At 7 dpi, when the neural induction was almost completed, most cells were positive for ZIKV E protein. Cells also expressed PAX6, a marker of the early ectoderm, thus indicating that ZIKV infection did not impair the differentiation process .Likewise, ZIKV replicated efficiently in NSCs infected prior to their differentiation into neurons. A progressive increase of ZIKV titer was measured in cell supernatant, followed by a decline due to massive cell death. Also in this case, ZIKV infection did not block the neural differentiation process, as shown by expression of the neuron-specific protein β-Tubulin in infected cells .Modeling of ZIKV infection during embryogenesis was performed by infection of hiPSCs with ZIKV MOI 1 during their differentiation into embryoid bodies (EBs), i.e., micro-tissue similar to native tissue elements. Viral infection was monitored by collecting the supernatants of infected cells and analyzing viral RNA copy number by qRT-PCR at different time points pi. Viral RNA load increased with time, reached a peak at 5 dpi and then decreased in parallel with the decrease of the number of live cells a. As shoIn the epidemiological context of the recent ZIKV epidemics in Asia and the Americas, the continuous expansion of WNV in Europe, and the dramatic increase of dengue epidemics, the aim of this study was to set up in vitro models of viral infection based on hiPSC-derived neural cells and to use these models to investigate and compare interaction of the three viruses with host human cells.Although hiPSC-derived NSCs, neurons and brain organoids have been extensively used to investigate ZIKV neural tropism and pathogenesis in comparison with other flaviviruses ,21,23,24In agreement with the accumulating evidences reporting infection of NSCs by ZIKV ,15,16,17DENV-2 and WNV also infected NSCs, hiPSCs, and neurons. DENV-2 infected hNSCs but at lower rate than ZIKV, in agreement with previous reports . At variBoth ZIKV and WNV exerted CPEs in hiPSCs, NSCs and neurons, leading to massive cell death, due in part to apoptosis mediated by activation of caspase-3, in agreement with previous reports ,29. At vRegarding the investigation of the innate antiviral response during ZIKV infection, studies based on different cell types and viral strains led to different observations. In primary human skin fibroblasts, ZIKV Asian lineage induced the upregulation of three important PRRs known to be activated by viral dsRNA, specifically TLR3, RIG-I, and MDA5, and, also, other factors involved in downstream pathways, such as IRF7, IFNα, IFNβ, and C-C motif chemokine ligand 5 (CCL5) . In the In this study, we investigated expression of a set of genes involved in antiviral innate immunity and showed that ZIKV, WNV, and DENV-2 infection induced a similar pattern of antiviral response in NSCs, neurons and hiPSCs, characterized by a marked upregulation of the dsRNA sensor MDA5 and the IFN-induced factors with antiviral activity IFIT1 and IFIT2. The highest expression levels of the antiviral genes were observed in cells infected by WNV, while ZIKV and DENV infections had milder effects on host innate antiviral response. These differences could be ascribed to the higher burden of WNV-infected cells due to higher infection and replication efficiency of the virus but also to the extent of viral inhibition of host innate immune response.IFIT2 in infected NSCs and activation of caspase-3.Previous studies demonstrated an antiviral function of IFIT2 in controlling viral replication in the CNS , and IFIMDA5, IFIT1, IFIT2, and other ISGs. Therefore, innate immunity represents an uncharacterized property of pluripotent stem cells that requires further studies to elucidate the molecular mechanisms.The literature reports absent or low levels of innate immune response against viral infection in mESCs , hESCs , and alsRecent reports demonstrated that ZIKV directly infects NSCs of the fetus and impairs growth in mice . ZIKV haIn conclusion, this study highlighted similarities and differences in ZIKV, WNV, and DENV mechanisms of infection of pluripotent stem cells and neural cells and induction of cell damage and antiviral response, which warrant further investigation. Several factors, both viral and cellular, are involved in the difference in infectivity, replication kinetics, and pattern of host antiviral response. These factors include, for example, expression levels of entry molecules on host cells and tissues and biding affinity with viral E proteins ,42. Othe5 cells were transduced with four Sendai virus vectors expressing the Oct4, Sox2, Klf4, and c-Myc, respectively, at a MOI of 10. Clones derived from reprogramming were manually picked, expanded, and stored in liquid nitrogen. Human iPSC clones were grown on irradiated mouse embryonic fibroblast  feeder layer with hiPSCs medium containing Dulbecco’s modified Eagle’s medium Nutrient Mixture F-12 (DMEM/F12), 20% Knockout serum replacement (KSR), 1% non-essential amino acids (NEAA), 1% GlutaMAX 100×, 1% Penicillin/Streptomycin, 1% of β-mercaptoethanol 1000×  and basic fibroblast growth factor  10 ng/mL; or onto feeder-free layer Geltrex substrate (Thermo Fisher Scientific) with serum-free StemMACS medium  supplemented with 1% penicillin/streptomycin (Thermo Fisher Scientific).Human iPSCs were generated by reprogramming of erythroblasts from healthy donors using Sendai virus-based vectors, as reported . Blood sNeural induction of hiPSCs was performed using Gibco PSC Neural Induction medium (Thermo Fisher Scientific) containing neurobasal medium and neural induction supplement 50×. Neural induction medium was changed every other day from day 0 to day 7 of neural induction. From day 7, primitive NSCs were maintained in NSCs expansion medium containing 50% neurobasal medium, 50% Advanced DMEM/F12 and neural induction supplement 50×, which was changed every other day.Expanded NSCs were differentiated into immature neurons. NSCs were seeded onto polyornithine- (20 µg/mL) and laminin- (10 µg/mL)  coated culture dishes in NSCs medium. The next day, NSCs medium was replaced with neural differentiation medium consisting of Neurobasal medium, 2% B-27 Supplement, GlutaMAX 100× 2mM and 1% penicillin/streptomycin . The culture medium was changed every 3–4 days.Embryoid bodies (EBs) formation was performed by plating hiPSCs in ultra-low-density attachment plates  in hiPSCs medium without bFGF. The medium was changed every 3 days. After 7 days, EBs were seeded on 0.1% gelatine-  coated 6-well tissue culture plate with DMEM, 10% FBS, 1% penicillin/streptomycin, 2 mM GlutaMAX (Thermo Fisher Scientific) and the medium was changed every 3 days. After 7 days of growth adhesion, cells were harvested using Trypsin enzyme (Thermo Fisher Scientific).hiPSC clones growing on MEFs were detached from the well with collagenase IV (Thermo Fisher Scientific) and plated in ultra-low-attachment plates (Corning Incorporated), in iPS medium without b-FGF. The medium was changed every three days; after seven days, EBs were transferred into a 0.1% gelatin pre-coated 6-well plate with DMEM 10% FBS 1% penicillin–streptomycin, 2 mM Glutamax .TUBB, PAX6), mesoderm , and endoderm .After a week of growth in adhesion, cells were harvested by trypsinization and total RNA was collected and reverse-transcribed into cDNA. Quantitative RT-PCR analysis was performed to assess the expression of the three germ layers markers: ectoderm , 1.7 × 107 pfu/mL (WNV), and 2 × 105 TCID50/mL (DENV-2).The following viral strains were used in experiments: ZIKV strain H/PF/2013 (GenBank KJ776791), Asian lineage, clinical isolate from French Polynesia, 2013; WNV strain AUT/2008 (GenBank KF179640), lineage 2, goshwak isolate from Austria, 2008; DENV serotype 2 strain, clinical isolate. Viruses were grown on Vero cells to generate viral stocks at titer of 1 × 102 for 24 h, cell growth medium was removed and replaced with infection medium, containing the virus diluted in DMEM with 1% penicillin/streptomycin (Thermo Fisher Scientific) at the specified multiplicity of infection (MOI). After incubation at 37 °C and 5% CO2 for 1 h and 30 min, the infection medium was replaced with growth medium and cells were maintained at 37 °C and 5% CO2. Mock-infected Vero cells were used as negative control in all infection experiments.Cells were seeded in tissue culture well plates; after incubation at 37 °C and 5% COViral replication kinetics was measured in cell culture supernatants and cell lysates collected at different time points pi. by using quantitative qRT-PCR with primers and TaqMan-probe sets specific for ZIKV NS5, WNV NS5, and DENV NS5, plaque count assay, and TCID50 assay.OCT4, NANOG, DNMT3B, TERT, REX1, and SOX2); the differentiation genes of the three germ layers , PECAM, CDH5, GATA2, FLK1 (mesoderm), AFP, GATA4 (endoderm)); the neural stem cell-specific genes NESTIN, PAX6, SOX1, SOX2 and the neuron-specific genes TUBB, MAP-2, NSE.RT-PCR was performed to evaluate the expression of the pluripotency genes  was analyzed by qRT-PCR analysis. Nucleic acids were purified from cells by using RNeasy Mini Kit . qRT-PCR was run on the 7900HT Fast Real-Time PCR System instrument (Thermo Fisher Scientific). qRT-PCR results were normalized to GAPDH mRNA and analyzed using the ΔΔCt method.Expression of genes encoding key factors involved in antiviral innate immunity  in phosphate-buffered saline , permeabilized with PBS/0.1% Triton X-100 (Sigma-Aldrich) and blocked in 4% bovine serum albumin (Sigma-Aldrich) in PBS. Then, cells were incubated with the primary antibodies diluted in PBS with 4% BSA. Primary antibodies specific for flavivirus E protein , PAX6 , Nestin , β-Tubulin , MAP-2 , OCT4 , SSEA3 and SSEA4 , KLF4 , SOX2  were used. All the antibodies were incubated overnight at 4 °C. The following secondary antibodies, diluted 1:250, were used: anti-mouse IgG FITC ; anti-rabbit IgG Alexa Fluor-546 . Nuclei were stained with DAPI  or DRAQ5 fluorescent probe solution (Thermo Fisher Scientific). Cells were visualized under a fluorescent microscope  with 20× magnification or a confocal microscope (Leica) with 63x magnification.4 cells/well). For each virus a MOI of 1 was adopted. Briefly, cells were detached from the well, fixed with 4% PFA  in PBS (Thermo Fisher Scientific), permeabilized in 90% methanol and stored at −20 °C overnight.The activity of Caspase 3 was measured at 72 hpi and 96 hpi in mock and infected NSCs plated in quadruplicate in 12 wells plates  diluted 1:100 in incubation buffer. After 1 h the antibody was removed and the cells were suspended in PBS for data acquisition with Becton Dickinson LSR II Flow Cytometer (BD Bioscience) and analysis using Flowing software.3 cells/well). Upon viral infection with a MOI of 1, cell viability was analyzed at 72 h and 96 hpi. Freshly dissolved solution of MTT  in PBS was added to each well and incubated for 4 h at 37 °C. Then, a solubilization solution (10% sodium dodecyl sulfate and 0.01 M HCl) was added and, after overnight incubation at 37 °C, absorbance was read at 620 nm.Cell survival was evaluated by MTT assay in mock and infected NSCs plated in 96 well tissue culture plates (8 × 10t-test and the statistical significance was defined as p < 0.05.Data were presented as mean value ± standard deviation. Statistical analysis was conducted using an unpaired Student’s