A new form-stable composite phase change material (PEG/ASB) composed of almond shell biochar (ASB) and polyethylene glycol (PEG) was produced via a simple and easy vacuum impregnation method. The supporting material ASB, which was cost effective, environmentally friendly, renewable and rich in appropriate pore structures, was produced from agricultural residues of almond shells by a simple pyrolysis method, and it was firstly used as the matrix of PEG. Different analysis techniques were applied to investigate the characteristics of PEG/ASB, including structural and thermal properties, and the interaction mechanism between ASB and PEG was studied. The thermogravimetric analysis (TGA) and thermal cycle tests demonstrated that PEG/ASB possessed favorable thermal stability. The differential scanning calorimetry (DSC) curves demonstrated that the capacities for latent heat storage of PEG/ASB were enhanced with increasing PEG weight percentage. Additionally, PEG/ASB had an excellent thermal conductivity of 0.402 W/mK, which was approximately 1.6 times higher than that of the pure PEG due to the addition of ASB. All the study results indicated that PEG/ASB had favorable phase change properties, which could be used for thermal energy storage. Energy demand and consumption are increasing noticeably and continuously due to the rapid development of society and the economy. Therefore, the methods used to promote energy utilization and conservation technologies have attracted great attention in various industries. Recent years have seen an increasingly intense research effort devoted to the study and application of phase change materials (PCMs), which show favorable abilities to store and release abundant thermal energy during the course of phase change ,2,3. PCMAn effective method to address these problems is to create form-stable composite phase change materials (CPCMs) that encapsulate PCMs by porous supporting materials, including graphene , diatomi4 impregnation. However, research reports about form-stable CPCMs based on biochar are still rare, and the use of biochar as the foundation of form-stable CPCMs production could not only solve the liquid leakage problem and improve the thermal conductivity of organic PCMs but could also make full use of agricultural solid wastes, reducing the preparation cost of form-stable CPCMs. Therefore, further studies of organic PCMs immobilizing in biochar are currently still required.Biochar, as a type of carbon-rich material, can be produced via pyrolysis treatment of biomass like agricultural or forests residues under an oxygen-limited environment ,24,25. CThus, in this work, the environment friendly, renewable and cost-effective biochar (ASB) with appropriate pore structures was prepared with agricultural residues of almond shells by a simple pyrolysis method, and then it was used as the matrix of PEG to develop a new form-stable composite phase change material (PEG/ASB) through a simple vacuum impregnation method. Different techniques were used to investigate the characteristics of PEG/ASB CPCMs such as structural properties and thermal properties. The study results suggested that PEG/ASB had favorable phase change properties, and that it could be used for thermal energy storage.1,a2), the almond shell biochar contained many particles of carbon with an abundance of pores. This structure suggested that ASB may have a considerable porosity and surface area, which may be beneficial for the adsorption and storage of PEG. Examination of 1,b2) shows that many of the ASB pores were filled, which meant that ASB adsorbed and fixed PEG successfully.The scanning electronic microscopy (SEM) images of ASB and PEG/ASB CPCM with 60% theoretical PEG content (CPCM3) are shown in 2/g, and the total pore volume was 0.17 cm3/g, suggesting that ASB may own favorable adsorption ability. It can be clearly seen from The pore size distribution curve for ASB is displayed in The leakage test results of PEG/ASB CPCMs are displayed in The X-ray diffraction (XRD) patterns of PEG, ASB and CPCM3 are displayed in −1 and 3446 cm−1 were observed due to the stretching vibrations of the C=C and –OH groups, respectively [2 functional groups were observed at 962 cm−1 and 2889 cm−1 [−1 and 3440 cm−1 [The fourier transform infrared (FT-IR) spectra of PEG, ASB and CPCM3 are displayed in ectively ,39,40. F889 cm−1 . Moreove440 cm−1 , respect440 cm−1 ,42, whicThermal stability is a significant property for the application of PCMs. The TGA and derivative weight loss (DTG) curves of PEG and CPCMs are displayed in As shown in The schematic illustration of the interaction between PEG and ASB is displayed in The phase change behaviors of PEG and PEG/ASB CPCMs, including the phase change temperatures and latent heats, were evaluated using the DSC technique. The DSC curves of PEG and CPCMs are displayed in The supercooling phenomenon of PEG and CPCMs can be analyzed via the test data obtained in DSC. As is shown in In this study, heat cycle experiments test of CPCM3 were used to evaluate the thermal reliability. The heat cycle tests were conducted using an oven at 65 °C, and the cycle index was 50. The DSC curves of CPCM3 before and after the heat cycle are displayed in The results of thermal conductivity tests of ASB, PEG and PEG/ASB CPCMs are shown in In this study, almond shells were obtained from apricots planted in Xinjiang, China. The clean almond shells were dried at 105 °C for over 24 h and were then crushed into powder by a rotary cutting mill with high speed. Then, the particles with diameters of under 200 meshes (≤0.074 mm) were collected for biochar production. The average molecular weight of polyethylene glycol (PEG) is 4000, and it was purchased from Shanghai Aladdin Bio-Chem Technology Co LTD . Absolute ethanol was purchased from Sinopharm Chemical Reagent Co LTD .2) flow was 50 mL/min. First, the heating process started at indoor temperature and was conducted at a heating rate of 10 °C/min until reaching the final pyrolysis temperature of 700 °C. Second, after this process continued for 2 h at 700 °C, the furnace was cooled down naturally to indoor temperature. Finally, the ASB lump was collected and ground into powder.The schematic illustration of ASB preparation is shown in The schematic illustration of PEG/ASB CPCMs preparation is shown in −1 to 4000 cm−1. The thermal properties of PEG and PEG/ASB CPCMs, including enthalpy and phase change temperatures, were obtained by differential scanning calorimetry . The samples were first heated from 0 °C to 100 °C and were then cooled from 100 °C to 0 °C at the rate of 10 °C/min rate under continuous nitrogen flow. The thermal stabilities of PEG and PEG/ASB CPCMs were analyzed by thermogravimetric analysis  from indoor temperature to 550 °C under a nitrogen atmosphere and with a heating rate of 10 °C /min. The thermal conductivity of PEG, ASB and PEG/ASB CPCMs were measured at 35 °C by a Xenon lamp flash thermal conductivity meter .The microstructure images of ASB and PEG/ASB CPCMs were obtained by scanning electronic microscopy . The Brunauer-Emmett-Teller  surface area and pore structure characteristics of ASB, including pore volume and diameter, were obtained via a nitrogen adsorption method. X-ray diffraction  patterns of PEG, ASB and PEG/ASB CPCMs were recorded at 40 kV and 30 mA at angles ranging from 10° to 60° to evaluate their crystal behaviors. The FT-IR spectra of PEG, ASB and PEG/ASB CPCMs were acquired by a Fourier transform infrared spectrometer , ranging from 400 cmIn summary, PEG/ASB form-stable CPCM was developed via the simple blending and vacuum impregnation method. The environmental and low cost ASB was produced from almond shells via a simple slow pyrolysis and it firstly acted as the supporting material of PEG. To study the characteristics of ASB, PEG and PEG/ASB, the structural properties were investigated by XRD, FT-IR, SEM and BET, and the thermal properties were examined using DSC, TGA and thermal conductivity test. The BET results indicated that ASB could be used as a good adsorbent material. PEG was fixed into ASB successfully, and there was no chemical reaction between PEG and ASB but rather physical interactions only. The latent heat storage capacities of the PEG/ASB were shown to increase as the PEG contents increased, and the prepared PEG/ASB possessed good thermal stabilities below 310 °C. The addition of ASB greatly improved the thermal conductivity of PEG/ASB compared to the pure PEG. In conclusion, the PEG/ASB could be used as a reliable energy storage material due to its good latent heat storage capacity and shape stability.


Segmental zoster paresis (SZP) of limbs, characterized by focal weakness of extremity, is recognized as a rare complication of herpes zoster (HZ). The following study analyzes the clinical characteristics and data from electromyography and MRI scans in patients with motor weakness after zoster infection.One thousand three hundred ninety-three patients from our database  suffering from HZ were retrospectively reviewed from June 2015 to July 2017. Patients who fulfilled the diagnostic criteria for SZP were included in the analysis. The clinical characteristics, as well as electromyography findings and MRI scans were analyzed.SZP was present in 0.57% of patients with HZ (8/1393). The average age of symptom onset in 8 SZP patients was 69 years old . The severity of muscle weakness ranged from mild to severe. The electrophysiological testing revealed the characteristics of axonopathy. Radiculopathy (2/8), plexopathy (2/8), radiculoplexopathy (3/8) and combined radiculopathy and mononeuropathy (1/8) were also identified. MRI revealed hyperintensity of the affected spinal dorsal horns, nerve roots or peripheral nerves.SZP is associated with obvious limb weakness, nerve axons lesions and localization to nerve roots, plexus or peripheral nerves. Herpes zoster (HZ) is caused by varicella-zoster virus (VZV) which is latent in the dorsal root ganglia and reactivates when the immune system is not functioning properly. The incidence of HZ is about 4–4.5 per 1000 person-years , 3.The exact mechanism of SZP is not clear, although the spread of virus along the nerve is presumed –6. The eHere, we investigated clinical, electrophysiological and imaging evidence in a case series of 8 patients with SZP, as well as possible factors influencing prognosis.The patient database in Shandong Provincial Qianfoshan Hospital was reviewed for coded diagnoses of HZ from June 2015 to July 2017. Patients with HZ were diagnosed as SZP based on the following criteria : 1) InfeWeakness was identified in each affected muscle as mild (corresponding to MRC 4 or 4+), moderate (corresponding to MRC 3), severe (corresponding to MRC 2 or 1) and complete (corresponding to MRC 0).According to the Medical Research Council scale, the muscle recovery was based on the following criteria: 1) complete recovery, if muscle strength was evaluated as grade 5 and the sensory symptom disappeared in the last follow-up visit; 2) no recovery, if no improvement in muscle strength was observed; 3) partial recovery, case between the complete and no recovery. In addition, all patients were followed up for approx. 0.5–2.0 years; the follow-up evaluation was based on sensorimotor symptom and muscle strength.This retrospective study was approved by the medical ethics committee at our hospital.S) and sensory nerve action potentials (SNAPS).Nihon Kohden MEB-9400 electromyograph was used to evaluate nerve injury. Surface electrodes were used to perform nerve conduction studies (NCS), including motor and sensory nerve conduction velocity (NCV), distal motor latency (DML), amplitudes of compound muscle action potentials  sides or normal values helped to determine the degree of damage. If NCV was slower than 75% of the lower limit of normal, and DML was longer than 130% of the upper limit of normal, it was regarded as demyelination. If the side-to-side amplitudes difference was greater than 50%, it was regarded as abnormal and axonal lesion.The needle electromyography (EMG) was performed by a concentric needle electrode, which was used to find spontaneous potentials such as positive sharp waves and fibrillation potentials conventionally graded from 0 to 4+ as follows: 0 for none present; + 1 for persistent single trains of potentials (> 2–3 s) in at least two areas; + 2 for moderate number of potentials in three or more areas; + 3 for many potentials in all areas; + 4 for full interference pattern of potentials. The firing pattern  was assessed.According to SNAP and spontaneous activities in paraspinal muscles, associated limb was characterized as preganglionic, postganglionic lesions or combined pre- and postganglionic lesions, and then further identified as radiculopathy, plexopathy, radiculoplexopathy and peripheral nerves  .The MRI results were reviewed by an experienced radiologist. All studies included both T1- and T2- weighted images in coronal planes, without gadolinium-enhanced T1-weighted images. All T2-weighted sequences had fat saturation or a short tau inversion recovery sequence. Nerve root, plexus, or peripheral nerve images were identified as abnormal if prolonged nerve T2 or nerve enlargement was shown based on comparison with contralateral neural structures within the imaging field.Eight out of 1393 inpatients with HZ fulfilled the diagnostic criteria for SZP, accounting for 0.57%, which was confirmed by neurologic examination and electrodiagnostic evaluation. Clinical characteristics are listed in Table Rash distribution in all patients corresponded to weakness distribution Fig. . AlthougFour patients had diabetes , while 1 patient had a 20-year history of rheumatoid arthritis and use of immunosuppressive medications (case 5). Antiviral drugs, such as acyclovir, were administered to 5 patients  orally or intravenously within 72 h after the rash appeared, 3 patients after 72 h. In addition, 4 patients received short term use of oral corticosteroids ; nevertheless, no patient experienced immediate pain or weakness relief after treatment.The prognosis was quite different Fig. . PatientS and SNAPS amplitudes of affected nerves, compared with the contralateral sides  in weak muscles. The positive sharp waves and fibrillation potentials in affected myotomes and the decreased recruitment of MUAPs suggested ongoing axonal lesions.S amplitudes, 2 among 8 were diagnosed with preganglionic lesions , 2/8 were characterized as having postganglionic locations  and 4 out of 8 were recognized as combined pre- and postganglionic lesions .According to spontaneous activities in paraspinal muscles and SNAPThe MRI examination was performed in 4 out of 8 patients no more than 1 week after electrophysiological testing , infraspinatus (1/5), supraspinatus (1/5) and biceps (2/5), according to the MRC scale. Distal muscle strength was normal. The right biceps reflex was absent. The electrophysiological examination revealed lower amplitude axillary and musculocutaneous CMAPs  compared to contralateral sides . The sensory nerve conduction studies were normal. Abnormal spontaneous potentials and decreased recruitments of MUAPs were present in the right deltoid, infraspinatus, biceps and C5–6 paraspinal muscles. In conclusion, the electrophysiologic findings were consistent with the incomplete lesions of C5 and C6 nerve roots.Within 3 months follow-up period, he regained the full arm strength without any treatments. Electrophysiologically, the amplitude axillary and musculocutaneous CMAPs  were normal. Abnormal spontaneous activities in muscles innervated by C5 and C6 nerve roots disappeared and many polyphasic MUAPs were observed.An 87-year-old woman developed burning pain and vesicular rash over the right lateral arm and forearm. Two weeks after rash, she was not able to elevate her right arm to the shoulder level, bend the forearm at the elbow joint or grip tightly. Moderate to severe weakness of C6–8 myotomes was observed, as well as the hypoesthesia over the C6–7 dermatomes. The biceps reflex was absent. The electrophysiological examination revealed decreased motor ampltitudes of axillary, musculocutaneous and median nerve  and the absence of median and radial nerve SNAPs. The needle EMG revealed many positive sharp waves in the right deltoid, biceps, extensor digitorum communis, abductor pollicis brevis and C6 paraspinal muscles. These findings were consistent with a right incomplete C6–8 radiculoplexopathy. Brachial plexus MRI showed hyperintensity of right brachial plexus especially at the C6–8 nerve roots level. Consequently, clinical one-year follow-up revealed that the patient was still not able to elevate her right arm to the shoulder level. Also, she presented with numbness of the thumb and post-herpetic neuralgia.A 61-year old man developed burning pain and vesicular rash over left thumb, index finger and forearm. Fifteen days after his rash, he noted weakness in his left hand dorsal stretch and grip. There was moderate weakness of muscles in left C6–8 myotomes and hypoesthesia over the thumb. The triceps muscle stretch reflex was absent. The electrophysiological examination revealed decreased amplitude radial CMAPs (4.1 mV) as compared to the contralateral side (11.1 mV) and decreased amplitudes median and radial nerve SNAPs. The needle EMG revealed many positive sharp waves in the left extensor digitorum communis, brachioradialis, abductor pollicis brevis and C7 paraspinal muscles. These findings were consistent with a left median and radial nerve lesions and nerve root lesion. Moreover, brachial plexus MRI showed hyperintensity of distal median and radial nerve. Though, there was no nerve enlargement or T2 hyperintensity of nerve roots. A half-year follow-up revealed that his muscle strength did not recover, and his muscles seemed atrophied. Also, he developed post-herpetic neuralgia.In the present study, we confirmed that varicella-zoster virus can lead to SZP which is an uncommon complication of a common ailment, including nerve roots, brachial or lumbar plexus, and peripheral nerves lesions. The electrophysiologic and imageological findings can help demonstrate the extent and severity of affected nerves or nerve roots.The elderly and immunocompromised patients are most susceptible to VZV , 9, 19. Yoleri et al. have suggested that zoster paresis preferentially strikes the upper limbs and then the lower limbs , while aOur electrophysiological study manifested low amplitudes of CMAPs or SNAPs together with many spontaneous activities in all patients, thus suggesting motor and sensory axonopathy. In axonal loss lesions, NCS and DML were normal or mildly slow. Generally, NCV was above 35 m/s and DML was shorter than 130% of the upper normal limit in axonal loss lesions. All our results from 8 patients met the criteria. Consequently, peripheral neuropathy caused by HZ with predominant axonopathy could be seen through our cases electrophysiologically. Sachs et al. have reported an electrophysiological study of zoster paresis for 22 months of follow-up, which appeared to be axonopathy and reinnervation .The dorsal root ganglion is the seat of VZV previous to the HZ eruption; therefore, it is no wonder that amplitudes of SNAPs were low, showing sensory neurons or axons damage. According to existing pathologic studies, demyelination, axon degeneration and lymphocyte infiltration can be found in affected nerves, dorsal root ganglions and dorsal horns . These fBrachial or lumbosacral plexus was affected in 5 out of 8 patients, suggesting the dorsal root ganglion (the seat of virus prior to its eruption) and ventral roots lesions. Although the precise mechanism of SZP remains unclear, it is easy to understand that the spread of VZV along nerve fibers to ventral root, ventral horn, and distal nerves can lead to corresponding lesions.Four of our patients underwent MRI examination. MRI is a useful tool for diagnosing SZP. The imaging abnormalities included nerve enlargement and T2 signal hyperintensity of dorsal horn and brachial plexus or peripheral nerves, which were consistent with clinical symptoms and elcetrophysiological findings, although these imaging manifestations are not specific and could be found in peripheral nerve inflammation caused by other diseases such as neurobrucellosis . The imaIn the literature, the prognosis for SZP is generally favorable, but the return of motor function can be incomplete, while recovery time can significantly vary , 32. TwoOur study is limited by a small sample size of this rare complication of HZ and retrospective design. Some patients without clinical zoster paresis were not examined with EMG, so there might be electrophysiological abnormalities in these patients. In addition, some patients were likely to have a longer follow-up for prognostic estimation.In summary, the present study highlights clinical, electrophysiological and imaging characteristics of this rare motor involvements of HZ. It is associated with significant limb weakness, obvious nerve axons lesion, and it is localized in nerve roots, plexus or peripheral nerves. The information is crucial for neurologists in order to avoid unnecessary misdiagnosis. Also, SZP should be considered in the differential diagnosis of acute painful motor weakness of limbs. The electrophysiological testing and MRI scan can be useful for confirming the diagnosis of SZP.


Syngnathus) and cod-like fishes (order Gadiformes) is of particular interest. The MHC II pathway is responsible for immunization and defence against extracellular threats through the presentation of exogenous peptides to T helper cells. Here, we demonstrate the absence of all genes encoding MHC II components  in the genome of an anglerfish, Lophius piscatorius, indicating loss of the MHC II pathway. By contrast, it has previously been reported that another anglerfish, Antennarius striatus, retains all MHC II genes, placing the loss of MHC II in the Lophius clade to their most recent common ancestor. In the three taxa where MHC II loss has occurred, the gene loss has been restricted to four or five core MHC II components, suggesting that, in teleosts, only these genes have functions that are restricted to the MHC II pathway.Genome studies in fish provide evidence for the adaptability of the vertebrate immune system, revealing alternative immune strategies. The reported absence of the major compatibility complex (MHC) class II pathway components in certain species of pipefish (genus  AlthougAn intact adaptive immune system has been found in almost all vertebrate species that have had their genomes sequenced, but recent work has demonstrated the loss of components of the adaptive immune system in the elephant shark, pipefish, coelacanth and the entire Gadiformes order –10. ThesThe teleost order Lophiiformes (Anglerfishes) harbours at least 321 living species, approximately half of which express some degree of sexual parasitism . In thesLophius piscatorius, and show that it has lost all components of the MHC II arm of the adaptive immune system. The MHC II pathway is known to be involved in allogenic rejection [Here, we present two independently obtained draft genomes of an anglerfish, ejection  and our 2.(a)L. piscatorius individuals (referred to as BF1 and BF2) were collected in the Bodø coastal waters, Nordland County in collaboration with local fishermen. BF1 skeletal muscle and BF2 kidney were used for subsequent total DNA isolation, library preparation and sequencing. BF1 total DNA sequencing was performed using Illumina MiSeq and SOLiD 5500 technologies (sequence depth: 24×). BF2 total DNA was sequenced by Dovetail Genomics, USA on an Illumina HiSeq X instrument (sequence depth: 150×) as a service. The Illumina libraries were 300 bp paired-end reads with 600 bp insert size for the MiSeq, and 150 bp paired-end reads with 350 bp insert size for the HiSeq.Samples from two (b)The raw reads were trimmed from adapters and low-quality bases using Cutadapt . Only IlMHC genes were identified using methods similar to those used in  figure . BrieflyL. piscatorius assemblies as well as on assemblies of Antennarius striatus, Gadus morhua and Perca fluviatilis [L. piscatorius, the unassembled reads (SOLiD and Illumina) were searched using tBLASTn. Matching reads were reassembled  and verified by reciprocal BLASTn against NCBI nr.We performed this analysis on our viatilis . If a geMore detailed descriptions are provided in the electronic supplementary material.3.(a)L. piscatorius assemblies contained 664 (BF1) and 724 (BF2) megabases with N50 values of 6.9 kb and 108 kb, respectively. We used the BUSCO [The resulting he BUSCO  actinoptA. striatus assembly . Hence, our assemblies are comparable to or better than assemblies in [The gene space completeness of our assemblies is thus similar to that obtained for the blies in  in terms(b)L. piscatorius as well as in species previously reported to either have  or lack (G. morhua) genes coding for MHC II components. Candidate orthologues were readily observed for all MHC I genes in all species. By contrast, we were unable to identify genes coding for CD4, CD74 A/B, MHC II α/β in either L. piscatorius or G. morhua assemblies .We used tBLASTn with a set of adaptive immune system genes to identify orthologous genes in semblies . We repelineages ; this toL. piscatorius we also searched for short sequences in the unassembled reads that could be aligned with the missing genes. Using tBLASTn, we identified 18 and 62 reads from BF1 and BF2, respectively, which aligned with an MHC II β subunit. To locate the position of these potential MHC II sequences, the matching reads were assembled into contigs and mapped back to the original assemblies. This identified a region of approximately 300–480 bp in length present in both assemblies. When translated, the predicted reading frame was interrupted by multiple stop codons , indicating that the fragment represents a remnant of an MHC II β chain gene. Hence, we conclude that the MHC II pathway is absent in L. piscatorius.To confirm the absence of MHC II orthologues in L. piscatorius, we identified contigs containing these genes [Gasterosteus aculeatus) loci . All highly conserved genes were identified in either a single (CD74) or three (CD4) contigs and the gene predictions lying within these contigs aligned both in terms of direction and order. CD74 in L. piscatorius seems to have been lost through a deletion of a region lying between ndst1a and SCL35A4 that has removed both CD74 and almost all intergenic space. For CD4, we were unable to identify a contig spanning the expected CD4 position; nevertheless, our analysis confirms the presence of the expected syntenic genes within our assembly.To confirm the presence of genes syntenic to CD4 and CD74 in se genes  and alig(c)Antennarius striatus has been reported to contain both MHC I and MHC II pathway genes [A. striatus and L. piscatorius are members of the Lophiiformes order, we considered the possibility that the identification of A. striatus MHC II orthologues could have resulted from cross-contamination of the sample. Although we did observe the presence of cross-contaminating mitochondrial sequences from distantly related teleost taxa, the relative sequencing depth of contaminant and A. striatus sequences, combined with the unimodal depth distribution preclude the possibility that the MHC II sequences were derived from contaminant DNA fragments . Our observations thus confirm the presence of MHC II, while at the same time highlighting the potential of cross-contamination leading to confounding results.ay genes . Since b4.Syngnathus typhle and S. scovelli) and now L. piscatorius lack a functional MHC II pathway [α and β chains . In addition, CD74 A/B has been completely lost in L. piscatorius and the Gadiformes order. Nevertheless, all contain a complete set of MHC I pathway components [α and β often occupy multiple loci on different chromosomes [A. striatus would require around 10 deletions , pipefishes ( pathway ,6,9,10. omosomes . This meeletions . This suBy contrast, genes such as CIITA, which is conventionally thought to be a specific activator of MHC II gene expression , is likeLophius species and has played an enabling role in the development of sexual parasitism.Sexual parasitism in anglerfish suggests some form of specialized immune system allowing for allogenic tolerance between fused individuals. Although CD8+ cytotoxic lymphocytes are thought to be the primary effectors of allogenic rejection, it is clear that MHC II components have both enabling and effector functions , and a lA. striatus clearly possesses all MHC II components. That would also mean that our observations are unlikely to be relevant to sexual parasitism in other anglerfish clades. However, inferences of higher-order taxonomies are still fraught with difficulty, exemplified by the fact that the phylogenetic topology of the taxa involved based on mitochondrial DNA is subject to change depending on the choice of outgroup [Most Lophiiformes phylogenies place the Lophioidei suborder at the most basal position in the anglerfish taxonomy, followed by Antennarioidei ,14,31,32outgroup ,13 (elec5.L. piscatorius and demonstrate a complete loss of the classical MHC II pathway in this species. The finding of a third taxon that lacks MHC II function corroborates the dispensability of MHC II in teleosts, and suggests that the genes lost in all three clades have no function outside of the MHC II.The classical MHC II components are responsible for the presentation of exogenous peptides to T helper cells and constitute an important part of the gnathostome adaptive immune system. Here, we report two draft genome assemblies of


MLH1, MSH2, MSH6 or PMS2. Carriers are predisposed to colorectal and endometrial cancer, but also other cancer types. The purpose of this retrospective study was to characterize the tumour spectrum of the Swedish Lynch syndrome families.Lynch Syndrome is an autosomal dominant cancer syndrome caused by pathogenic germ-line variants in one of the DNA-mismatch-repair (MMR) genes Data were obtained from genetically verified 235 Lynch families from five of the six health care regions in Sweden. The material was stratified for gender, primary cancer, age and mutated gene and the relative proportions of specific cancer types were compared to those in the general population.MLH1 and MSH2 comprised 78% of the cohort. Among the 482 non-colorectal/non-endometrial cancer diagnoses, MSH2 carriers demonstrated a significantly increased proportion of urinary tract, gastric, small bowel, ovarian and non-melanoma skin cancer compared to the normal population. MLH1 carriers had an elevated proportion of gastrointestinal cancers , while MSH6 carriers had more ovarian cancer than expected. Gastric cancer was predominantly noted in older generations.A total of 1053 family members had 1493 cancer diagnoses of which 1011 were colorectal or endometrial cancer. Individuals with pathogenic variants in MSH2-carriers. Gender and age also affect the tumour spectrum, demonstrating the importance of additional environmental and constitutional parameters in determining the predisposition for different cancer types.Lynch syndrome confers an increased risk for multiple cancers other than colorectal and endometrial cancer. The proportions of other cancers vary between different MMR genes, with highest frequency in  MLH1, MSH2, MSH6 or PMS2 [MLH1 and MSH2, which might reflect differences in expression between different pathogenic MMR variants, such as later age at onset of disease and reduced penetrance suggested in MSH6 and PMS2 [Lynch syndrome (LS) is an inherited autosomal dominant condition predisposing mainly to colorectal and endometrial cancer . In addi or PMS2 , 5, 7. Mand PMS2 .LS should be suspected in patients with an age onset of colorectal cancer before 50 years, proximal localisation of colorectal cancer, high DNA microsatellite instability in histological specimen of colorectal cancer, multiple primary meta/synchronous polyps/tumours in colon, rectum and endometrium, endometrial cancer before 50 years of age, or a familial clustering of cancer diagnoses, using criteria such as Amsterdam II or Bethesda guidelines , 10. SurThe study was approved by the Regional Ethical Review Board in Stockholm, Sweden. In Sweden, patients with a family history suggestive of LS are generally referred for genetic counselling to a department of clinical genetics at six university hospitals that provide regional genetic services with family investigations, genetic testing and recommendations for surveillance. For our study, five of six nationwide genetic centres in Stockholm, Uppsala, Umeå, Linköping and Lund  agreed to participate, providing us with anonymous full pedigree information. The information regarding cancer diagnosis, age at onset, or date of death were confirmed by medical reports or death certificates when available with the written consent from the affected relative, or (if deceased) from the closest relatives. All pedigrees harboured at least 3 consecutive generations and contained information about gender, type of gene variant, birth date, age at cancer diagnosis, cancer according to the ICD7 classification, tumour site and age at death and the status of pedigrees has been updated as of December 2014.Patients with early onset LS spectrum early onset cancer, synchronous or metachronous cancer, or Amsterdam II and/or Bethesda criteria fulfilled were genetically tested for LS. Genetic screening of the affected family members was performed in most cases using mainly Sanger DNA sequencing or otherwise massive parallel sequencing. The sequencing analyses were combined with multiplex ligation-dependent probe amplification  for the detection of large deletions or duplications.Statistical analyses included family members with a proven pathogenic germ-line variant, obligate carriers, individuals with a 50% risk of having a pathogenic variant, or combinations thereof. Obligate carrier status was allocated to members due to their position in the pedigree in relation to relatives with known pathogenic variants or other obligate carriers. First degree relatives to proven or obligate carriers who had not been tested for the familial variant were assigned a 50% carrier probability. These 265 individuals with cancer increased the number of tumours in the analysis, but due to a potential risk of error as their genetic status was unknown, we redid the analysis only including those individuals with known genetic status with similar results (data not shown).Analysis of the relative proportions of cancer diagnoses was performed as previously described . The age. In 1053 family members, at least one cancer diagnosis was identified  was too low for further analysis.As the spectrum of LS associated tumours is influenced by the genetic composition of the cohorts, we stratified the study population according to mutated MMR gene. We could not analyse MLH1 carriers had an elevated frequency of gastric, pancreas and small bowel but not of skin, urinary tract or ovarian cancer (Table MLH1 cohort was a few years younger than the average age in the population (Swedish Cancer Registry).er Table . The meaMSH2 carriers had an elevated proportion of several cancers including urinary tract, gastric, non-melanoma skin, ovarian and small bowel cancer (Table MSH2 carriers diagnosed with urinary tract and ovarian cancer showed a greater proportion with onset before age of 50 years in comparison with MLH1 carriers (Table MSH2 carriers where 34% were diagnosed before 40 years (data not shown). Of note, the elevated proportion of non-melanoma skin cancer found in MSH2 carriers  occurred before age of 50 years (Table In the group with pathogenic variants in p Tables . The pront Table . Of 8 can = 0), skin cancer (n = 1) and urinary tract cancer (n = 10) was too limited for analysis in the MSH6 group.The number of affected individuals with small bowel cancer  in 200 asymptomatic LS family members. No cases of small bowel cancer could be detected in the study group during the 2-year surveillance [Small bowel cancer is a rarity, representing 0.5% of cancer cases in Sweden 2010, while the cumulative lifetime risk in the LS group has been estimated to be between 0.6–7% , 5. The eillance . Recentleillance . Based oMSH2 and MLH1 carriers, as the number of families with pathogenic variants in MSH6 have been too low in most studies for conclusive results [MSH6 carriers indicate a 10–13% cumulative risk for ovarian cancer, comparable to that of MSH2 carriers and perhaps higher than the risk for MLH1 carriers [MSH2 and MSH6 carriers demonstrating an increased frequency of ovarian cancer, while the MLH1 carriers did not. A striking observation in our study was the high proportion of ovarian cancer before the age of 50 years in mutation carriers: 80% for MSH2, 63% for MLH1 and 42% for MSH6  will be missed. Inaccessibility to older medical reports was another concern, preventing the verification of cancer diagnosis in around 10% of the older generations in our study cohort. The retrospective nature of our study and the paucity of individuals with pathogenic variants in PMS2 and MSH6 necessitated wide confidence intervals that may have led to an underestimation of cancer types in these two groups. In addition, in former generations LS patients often died from their first cancer, as opposed to today when most LS patients under surveillance survive their first as well as subsequent cancers and thus may develop other late-onset tumours not seen in previous generations. This may bias our results.Our material is based on recruitment of patients with colon cancer and/or endometrial cancer, through the Swedish Departments of Oncogenetics and usually there is early onset of cancer or clustering of several cancer diagnoses in the family. This may have led to selection bias, e.g. cases with pathogenic MMR variants of low penetrance . In addition, pedigrees were comparably vast, containing at least three consecutive generations. Furthermore, the size of our cohort is not negligible covering a total of 1053 LS patients with cancer. Another strength of our study was the confirmation of clinical data  in 90% of our LS cohort through the Swedish cancer registry.MSH2 carriers develop multiple other cancers including gastric, urinary tract, ovarian, small bowel and non-melanoma skin cancer. In contrast, MLH1 carriers show an increased proportion of gastrointestinal cancers and MSH6 carriers of ovarian cancer. In addition, gender affected the tumour spectrum, with non-melanoma skin cancer noted in women only. The tumour spectrum also varies between genders and as over time, demonstrating the importance of not only genetic but also environmental factors in determining cancer predisposition. Our results may contribute to more accurate cancer risk estimations in Lynch syndrome patients thus providing better evidence upon which to base surveillance recommendations.In summary, the tumour spectrum of Swedish Lynch syndrome patients overlaps with that of LS patients in other Western countries. In addition to the increased risk of colon and endometrial cancer,


Endothelial cells (ECs) could express some important cytokines and signal molecules which play a key role in normal hematopoiesis and repopulation. Busulfan-induced vascular endothelial injury is an important feature after hematopoietic stem cell transplantation (HSCT). But the molecular mechanism of how the injured ECs affect hematopoietic reconstruction is still unknown. It is possibly through modulation of the change of some gene expression. RT-qPCR is one of the most popular methods used to accurately determine gene expression levels, based on stable reference gene (RG) selection from housekeeping genes. So our aim is to select stable RGs for more accurate measures of mRNA levels during Busulfan-induced vascular endothelial injury. In this study, 14 RGs were selected to investigate their expression stability in ECs during 72 hours of EC injury treated with Busulfan. Our results revealed extreme variation in RG stability compared by five statistical algorithms. ywhaz and alas1 were recognized as the two idlest RGs on account of the final ranking, while the two most usually used RGs (gapdh and actb) were not the most stable RGs. Next, these data were verified by testing signalling pathway genes ctnnb1, robo4, and notch1 based on the above four genes ywha, alas1, gapdh, and actb. It shows that the normalization of mRNA expression data using unstable RGs greatly affects gene fold change, which means the reliability of the biological conclusions is questionable. Based on the best RGs used, we also found that robo4 is significantly overexpressed in Busulfan-impaired ECs. In conclusion, our data reaffirms the importance of RGs selection for the valid analysis of gene expression in Busulfan-impaired ECs. And it also provides very useful guidance and basis for more accurate differential expression gene screening and future expanding biomolecule study of different drugs such as cyclophosphamide and fludarabine-injured ECs. The vascular endothelial cells (ECs) are particularly vulnerable to toxic effect of preparative regimen drugs, such as Busulfan, cyclophosphamide, and fludarabine which are widely used prior to hematopoietic stem cell transplantation (HSCT) . SeveralBusulfan, most widely used in HSCT, has been identified as having potent antitumor activity and inhibitory functions on normal hematopoiesis as well as myelogenous proliferation . Most imReverse-transcription-qPCR is one of the most widely used methods directly evolved from the end-point detection PCR to detect gene expression level under different research conditions because of its time-saving, high sensitivity, and specificity –14. But For the biomolecule study of Busulfan on EC injury, identifying the most stable RGs in Busulfan-impaired EC system firstly is of great importance. But, based on our knowledge, stable HKGs selection in the damaged ECs has never been performed. So the purpose of this research is to recognize the most suitable HKGs in impaired ECs, which can be used as reference genes for normalization of qPCR results.In the this study we used three software types including geNorm, NormFinder, and BestKeeper together with the delta-delta method –24 and C2, respectively. The medium containing DMEM, 10% FBS, and 30mg/L of Busulfan (purchased from Sigma-Aldrich) was added to the ECs and then observed at different time interval at 0, 12, 24, 36, 48, and 72 hours, respectively. All our samples were independently prepared three times.The endothelial cells (bEnd.3) were purchased from the Global Bioresource Center of American Type Culture Collection (ATCC) and cultured in a medium which comprised DMEM and 10% FBS and then incubated at 37°C in incubator and humidified at 5% CORNA was isolated from the normal and Busulfan-injured ECs using TRIZOL Reagent, Invitrogen kit according to the manufacturer's instructions . The conμm oligonucleotide dT primer; 10mM dNTP and DEPC-treated water were combined together and preserved at 65°C for 10 minutes with extended temperature of 4°C in the conventional PCR. The transcription mixture of 0.1M DTT, 50,000U M-mlv, and 5x-strand buffer was then incubated at 37°C for 50 minutes, at 70°C for 5 minutes and extended temperature of 4°C. All cDNA was synthesized from 300ng isolated RNA sample in a total volume of 20μL and kept at -20°C until ready for use. All samples were diluted by 1:10 with DEPC water, in order to achieve equal concentration of our samples for RT-qPCR analysis.The intact RNA was reverse transcriptase at once after isolation using Invitrogen reagent M-MLV. Firstly, the RNA was transcribed into first cDNA, using 10μL reaction contained 10μL of 2× Supermix SYBR Green I Master, 1μL forward and reversed primers, 8μL of distilled water, and 1μL of cDNA. The RT-qPCR program was comprised of predenaturation at 95°C for 1 minute, 40 cycles at 95°C for 20 seconds, 60°C for 15 seconds, and 72°C for 15 seconds.The 14 candidate reference genes primers and three target genes  were designed and purchased from Thermo Scientific and were selected in consideration of different intracellular biological function as shown in (−1/A)∗100] of RT-qPCR. The standard curve was obtained by plotting the Cp value (y axis) against the logarithm of the total cDNA concentration (x axis).The specificity of the primers and the size of the PCR products were checked using 2% agarose gel electrophoresis and gel-red staining. The threshold values  were obtained using a fluorescence threshold of 1.0. Then the results were copied into input file, based on the software requirement. The efficiency of each primer was calculated using the standard gradient , while those with higher value  had more variation in gene expression. The Pearson coefficient of correlation (r2/V3) of this dataset would be adequate for accurate normalization as shown in geNorm software grades the stability of the candidate genes based on the calculated M value. Genes with lower M value are considered as the most stable genes, while those with higher M value are the least stable genes. The three most stable genes include ywhaz and hprt1 both with an M value of 0.227 and alas1 with an M value of 0.284. The three reference genes with the highest M values were ubc (M = 0.962), tbp (M= 0.883), and rplo (M= 0.799), considered to be least stable expressed genes . In addiThis software determined the stability value of the selected references and at the end it identified the best two combinations of genes. Genes with the lowest stability values are considered as the most stable , while those with the higher stability values are the least stable genes . The two best combined genes revealed by the software include hmbs and gusb with a stability value of 0.081 as shown in  CT method determines the stability of housekeeping genes based on relative expression comparison within the samples. The lower ΔCp value between samples of gene tested is considered to remain constant. In order to obtain the most stable gene, the lower ΔCp value is deducted from other Cp values [The ΔΔp values , 44. TheIt illustrated the calculations of standard deviation (SD), variance, and mean fold difference using the delta-delta method and true fold difference determined by using log base 2 power of delta-delta value to determine the actual fold difference between the selected reference genes.We coranked the four methods to determine the most stable RGs in our study. In order to achieve the overall ranking, we allocate a weight to all the genes, which is then used to calculate the geometric mean . The geoTo evaluate the performance of the selected reference genes, we detect three target genes from three different signalling pathways and normalized the gene expression level or the fold change values using different selected references genes. We selected the best two candidate genes  and the two most often used RGs (actb and gapdh). The results are shown in When ywhaz was used as reference gene, we also found that robo4 expression level was significantly increased from 12h (0.7±0.3) to 48h (8.0±1.0) as shown in α in relation to gene expression [st, hmbs 3rd, actb 7th, tfrc 8th, gapdh11th, and rplo 9th, respectively. These findings suggested that it is very important to assess the expression stability of the selected RGs in any experimental research for ECs.The ECs have been suggested recently to play an active role in normal hematopoiesis and HSC trafficking , 8. Meanpression . Howeverpression  and the pression . Interes-ΔΔCt, and Comprehensive Ranking methods). The pairwise comparison approach includes the BestKeeper and geNorm, and this software identifies the most stable RG based on the expression of variation ratios among the RGs in our sample collected. The ratio variation (pairwise variation) for two candidate RGs across the sample measures the stability of the gene. However, the stability measurements of all the methods were combined to obtain the overall ranking of selected genes. The grading of RGs stability may be different due to the discrepancies of the program software. For example, the geNorm software ranked ywhaz and hprt 1 as the two most stable genes, while the NormFinder ranked the hmbs and gusb as the most stable genes. On the other hand, the BestKeeper ranked ywhaz and actb as the two most stable genes, while 2-ΔΔCt method ranked alasi and hprt1 as the two most stable genes. So, finally, an overall ranking is established by calculating the geometric mean in order to minimize the differences between the statistical programs  and delta-delta method and have a final consensus of the most stable RGs. Based on the overall ranking, ywhaz was graded as the most stable among the 13 HKGs, together with alas1 and hmbs, respectively. These three genes were considered as the most appropriate HKGs because they displayed minimal fluctuations under our experimental conditions.In our study we employed five methods (geNorm, NormFinder, BestKeeper, 2Some previous reports have suggested that accurate measurement of expression levels of normalization required multiple HKGs . Here weOur study demonstrated that Busulfan can strongly influence the stability of RGs. ywhaz and alas1 but not actb or gapdh were recognized as the idlest RGs for normal and injured ECs. This will be helpful for detection of gene expression in relation to both normal and Busulfan-injured ECs. Moreover, we also found that robo4 was significantly upregulated in Busulfan-injured ECs with time prolonged by optimized gene expression analysis. In conclusion, the results showed strong difference in gene expression level of three target genes using four different RGs which means that RGs selection is necessary for RT-qPCR normalization.


Sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affect small ruminants and cattle causing sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) respectively. In endemic areas, vaccination with live attenuated vaccines derived from SPPV, GTPV or LSDV provides protection from SPP and GTP. As live poxviruses may cause adverse reactions in vaccinated animals, it is imperative to develop new diagnostic tools for the differentiation of SPPV field strains from attenuated vaccine strains. Within the capripoxvirus (CaPV) homolog of the variola virus B22R gene, we identified a unique region in SPPV vaccines with two deletions of 21 and 27 nucleotides and developed a High-Resolution Melting (HRM)-based assay. The HRM assay produces four distinct melting peaks, enabling the differentiation between SPPV vaccines, SPPV field isolates, GTPV and LSDV. This HRM assay is sensitive, specific, and provides a cost-effective means for the detection and classification of CaPVs and the differentiation of SPPV vaccines from SPPV field isolates. The responsible viruses, sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) are large, complex, double-stranded DNA viruses of the genus Capripoxvirus, subfamily Chordopoxvirinae, family Poxviridae3. Due to the economic importance of the cattle, sheep and goat farming industry and the viruses potential for rapid transboundary spread, SPP, GTP and LSD are categorized by the World Organisation for Animal Health (OIE) as notifiable diseases5.Sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) are three important pox diseases of sheep, goat and cattle respectively7. SPP incursions have also been reported in Bulgaria and Greece7. In early 2018, outbreaks of SPP in Greece went uncontrolled despite implementation of an extensive stamping out campaign7. According to the World Animal Health Information System (WAHIS) web portal[WAHID]8, the disease is also present in Russia with several sporadic outbreaks reported between 2008 and 2019. The endemic geographic range of LSD was limited to the African continent including Madagascar9 until recently when the disease emerged in the Middle East before spreading into Asia and Europe. Outbreaks in Albania, Bulgaria, Greece, the Former Yugoslav Republic of Macedonia, Montenegro, Serbia, and the Russian Federation, increase the disease risk for the neighboring countries in the region10.SPP and GTP are endemic in Africa, except southern Africa, across the Middle East and Asia12. As live poxviruses may cause adverse reactions in vaccinated animals, it is crucial to use diagnostic tools that can rapidly and specifically differentiate virulent SPPV field isolates from attenuated SPPV vaccine strains. This would help  to elucidate the origin of disease in the event of mild or systemic post-vaccination reactions in vaccinated animals13. Similar assays have proven to be very useful in cattle to differentiate the LSDV Neethling vaccine from LSDV fields isolates in Israel14, Greece15 and Russia16.Live attenuated SPPV, GTPV and LSDV derived vaccines are widely used in many endemic African, Middle Eastern and Asian countries to control SPP and GTP17, or the genotyping of CaPVs20, however, a single assay to perform both methods has not been described. This can be accomplished by identifying a suitable genetic marker in the CaPV genome that will allow for the development of a unique, cost-effective test that can be used for both outbreak sample screening and epidemiological investigations in previously vaccinated herds.Routine capripox diagnosis would be improved if one assay was designed to provide both differentiation of SPPV field isolates from SPPV vaccine strains and simultaneously classify the sample into one of three known species: SPPV, GTPV and LSDV. Several tests have addressed the differentiation of SPPV vaccine strains from SPPV field isolates24, including the differentiation of vaccine strains from field isolates16.With the new generation of dyes and technological improvements in qPCR platforms, high-resolution melting (HRM) curve analysis has become a powerful technique for genotyping microbes, offering the possibility of developing methods other than sequencing for rapid pathogen typingIn this paper, we describe the identification of a novel target in the capripox genome and the development of an HRM curve analysis to differentiate SPPV vaccine strains from SPPV field strains and simultaneously identify the three species of CaPVs to support epidemiological surveillance and disease diagnosis during the vaccination period.The analysis of multiple-sequence alignments of CaPV genomes, including a SPPV vaccine from Morocco, enabled the identification of a region with a 21-nucleotide and 27-nucleotide deletion, unique to the SPPV vaccine. This region, within the CaPV homolog of the variola virus B22R gene, also harbored specific nucleotide differences between SPPV field isolates, GTPV and LSDV Fig. . The seqThis region was then targeted to design another set of primers for the amplification of a 110 bp fragment in SPPV vaccine strains and a 158 bp fragment in SPPV field strains, GTPV and LSDV.Subsequently, we tested the expected melting profiles and melting peaks of the predicted amplicons for SPPV vaccine strains, SPPV field isolates, GTPVs and LSDVs using the uMelt software. The results show four different and distinct melting peaks for SPPV vaccine strains, SPPV field isolates, GTPVs and LSDVs  were 76.90 ± 0.13 for SPPV vaccine, 80.27 ± 0.11 for SPPV field isolates, 81.58 ± 0.06 for GTPV and 82.29 ± 0.07 for LSDV.Following an initial testing of a panel of primers, the pair of forward and reverse primers indicated in Table To enhance and clarify the data analysis, we used the HRM software to further analyze the melting curves of the PCR amplicons. The HRM analysis of the normalized melting Fig.  and diffThe limits of detection, with ≥95% confidence limits, were: 38.04 (31.1–54.14), 17.80 (14.62–26.46), 16.27 (12.63–25.53) and 17.01 (13.22–26.64) copies per reaction for SPPV vaccine, SPPV field isolates, GTPV and LSDV respectively.To study the discriminating power of the HRM assay, 61 DNA samples extracted from SPPV vaccines, cell culture supernatants and clinical samples of CaPVs field isolates, from various geographical locations were screened  to analyze the potential of the assay to detect coinfection of SPPV vaccine with SPPV field isolates or GTPV or LSDV. At all proportions tested, we observed two readily distinct peaks for the mixture SPPV vaccine/GTPV (at Tm 76.26 for SPPV vaccine and 81.58 for GTPV), and the mixture SPPV vaccine/LSDV (Tm 76.58 for SPPV vaccine and 82.36 for LSDV). In contrast, for the mixture SPPV vaccine/SPPV field produced two readily distinct peaks (Tm 76.38 for SPPV vaccine and 80.25 for SPPV field isolates) at the proportion of 9/1 only. The mixture at the proportion 8/2 presented only a shoulder peak at Tm 76.38 (for the vaccine) and the SPPV field peak at Tm 80.25. The SPPV vaccine peak was absent at other proportions of the mixtures SPPV vaccine/SPPV field. Overall, there was a small shift in the Tm of SPPV vaccine in the mixtures as compared to SPPV vaccine tested alone.We also tested artificial mixtures of control plasmids at 10The specificity of the assay was evaluated by testing 13 DNA extracts from other ruminant pathogens , QuantStudio 6 (LifeTechnologies), RotorGene Q (Qiagen) and LightCycler 480 (Roche). This assay was successfully run on all four platforms, utilizing the same mixture and protocol, and succeeded in differentiating SPPV field isolates from SPPV vaccine strains as well as placing the samples in the correct genotypes. , a vaccine strain comparable to those tested in this study.Analysis of the partial fragment of the B22R gene in 37 representative SPPV vaccines, SPPV field isolates, GTPVs and LSDVs available for this study, confirmed two sets of deletions (21 bp and 27 bp) that are specific to the SPPV Romanian and Yugoslavian RM65 vaccine strains, which are used in Morocco, Egypt, Senegal and Algeria and the Romanian Fenner strain (MG000157), which is used as a SPPV vaccine in India. However, the comparisons showed that these deletions are absent in the B22R gene of SPPV NISKHI, an SPPV vaccine used mainly in Russia and countries of the former Soviet Union such as Kazakhstan20, for all 61 samples in this study. Of interest, is that the assay identified and correctly assigned previously characterized isolates into the correct CaPV species20: SPPV KS1, collected from sheep, was identified as a LSDV; SPPV Oman, GTPV Kitengela/O58/2011 and GTPV Kitengela/O59/2011 collected from sheep were identified as a GTPVs; GTPV Saudi Arabia, collected from a goat was identified as a SPPV. Similarly, two recent isolates, GTPV_Awi/O13/2011 and GTPV_Bale/O14/2007, both collected from sheep were identified as GTPVs.The comparative analysis of the partial B22R sequences of SPPV field isolates, GTPVs and LSDVs generated in this study with those retrieved from full genomes in public databases also confirmed the presence of species-specific signatures that were well-conserved within each of these 3 species, with minor sequence variability noted for both SPPV field isolates and GTPVs , Greece (KY829023), Serbia (KY702007) . To broaden the scope of the assay to enable the classification of CaPVs, we selected the final region based on the following: (1) the selected region offered markers for an unequivocal differentiation of SPPV vaccines, derived from the Romanian and Yugoslavian RM65, from SPPV field isolates; (2) the region presented enough nucleotide differences between the SPPVs, GTPVs and LSDVs that enabled the separation of these three species by HRM; (3) the differences between SPPV, GTPV and LSDV were well conserved within each CaPV species, thus representing strong species-specific signatures for genotyping. A region within the B22R gene of CaPVs which contains two separate nucleotide deletions of 21 and 27 nucleotides in length was selected. The same region also presented species-specific signatures for SPPV, GTPV and LSDV. A primer pair , synthesized and purified by reverse phase high-performance liquid chromatography by Eurofins, Germany. The specificity of the primer sequences were checked using the Basic Local Alignment Search Tool (NCBI/Primer-BLAST). From now on, we will refer to each of these four groups, SPPV vaccines, SPPV field isolates, GTPVs and LSDVs, as genotypes.In order to identify regions with unique differences between SPPV vaccines and SPPV field isolates, we scanned the sequence alignments of a draft full genome SPPV vaccine from Morocco and representative field isolates and vaccines of all three CaPV species. The sequences were retrieved from Genbank  as per the manufacturer’s instructions. The ligated products were used to transform E. coli DH5α competent cells . The positive clones were propagated, and the plasmid DNA extracted using the PureYield plasmid Midiprep System . The purified plasmids were quantified with the Nanodrop 3300 Fluorospectrometer using the PicoGreen dsDNA reagent kit  per manufacturer’s instructions and sequenced to confirm the presence of the correct insert. The copy number was calculated as described by Lamien The 10 µl PCR reaction mixture contained 1X Lightscanner master mix , 250 nM of each primer (Cap_ B22RDIV_For and Cap_ B22RDIV_Rev) and 2 µl of sample DNA. The PCR reaction was performed in a LightCycler 480 II Real time PCR Detection System  with an initial denaturation at 95 °C for 4 min, followed by 42 cycles of 95 °C for 5 sec, 58 °C for 5 sec and 72 °C for 5 sec. The PCR products were then denatured at 95 °C for 30 sec, cooled to 65 °C for 1 min, and melted from 65 °C to 90 °C at a rate of 100 acquisitions per °C. For each set of reactions, positive control plasmids and negative controls consisting of nuclease-free water in place of the template DNA were included. High Resolution Melting (HRM) curve analysis was performed using the LightCycler 480 Gene Scanning Software (Roche) to analyze the data and melting profiles of SPPV vaccines, SPPV field isolates, GTPVs and LSDVs. Normalized melt curves and difference plots were obtained by analyzing the active melt region and designating the pre and post melt regions.7 to 101 viral copies/µL using Herring sperm DNA matrix (5 ng/mL) and kept at 4 °C until further analysis. First, the linearity of the assay was analyzed for each viral species to determine the efficiency and dynamic range of the assay. Subsequently, the limit of detection (LOD) of the method was assessed by amplifying eight different concentrations  of each plasmid, corresponding to each of SPPV vaccine, SPPV field isolate, GTPV and LSDV. Five replicates of different plasmid concentrations were tested on four separated occasions, and the proportion of positive results were determined for each concentration. The LOD of the current assay was determined by probit regression analysis.Each plasmid was 10-fold serially diluted from 10The discriminating power of the developed HRM assay was evaluated using DNA (n = 61) extracted from suspected capripox clinical samples and CaPV infected cell culture suspensions from sheep, goats and cattle from different geographical regions  via Rstudio (version 1.1.383). A One-Way ANOVA test and Tukey multiple comparisons of means were performed in R to determine whether the average Tm differences between the genotypes were significant. In addition, box and whisker plots were constructed to illustrate the differences between the Tm of the four genotypes using the ggplot2 package in R. For LOD determination, the LC_probit function of the ecotox package in R was used to calculate the predicted limits of detection and their confidence limits (CL) using a probit analysis. The dose-response curves were plotted using ggplot2 in R.The cross-platform compatibility of this assay was evaluated using various real time PCR instruments. Thus, using the same PCR mix and protocol as in the LightCycler 480 II (Roche) instrument, PCR and melting curve acquisition analysis was also conducted using the CFX96 Real Time PCR system , QuantStudio 6 Flex Real Time PCR system , and Rotor Gene Q Real Time PCR cycler (Qiagen).A 250 bp region of the B22R gene containing the targeted HRM fragment was amplified by PCR for selected samples, using the primers B22R seqHRM-For and B22R- seqHRM-Rev Table , and seqSupplementary file


Aedes aegypti, since 2009, when autochthonous transmission of the virus was first recorded in the city. The aim of this work is to characterize the emergence of dengue and related arboviruses (Zika and chikungunya) in Córdoba since 2009. Herein, we present a data set with all known information about transmission of dengue, Zika, and chikungunya viruses in Córdoba, Argentina from 2009–2018, including what information is known of dengue virus (DENV) serotypes in circulation and origins of imported cases. The data presented in this work will assist researchers in investigating drivers of arbovirus emergence and transmission in Córdoba, Argentina and contribute to a better understanding of the global problem of the expanding distribution of arbovirus disease transmission.The distribution of arbovirus disease transmission is expanding from the tropics and subtropics into temperate regions worldwide. The temperate city of Córdoba, Argentina has been experiencing the emergence of dengue virus, transmitted by the mosquito  Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.10120445 Aedes aegypti mosquito control programs aimed at eliminating Yellow Fever in prior decades1. Dengue fever is caused by dengue virus (DENV serotypes 1–4), which causes a spectrum of acute febrile illness1. In Argentina, dengue was reported for the first time in over 80 years in the northwestern Province of Salta in 1997, and has since been largely constrained to northern provinces of the country that have a subtropical climate3.Dengue fever re-emerged in Latin America and the Caribbean in the 1980s, following the decline of widespread 4, fourteen years after Ae. aegypti was first detected in the city5. Since that time, Córdoba has reported imported and autochthonous cases of Aedes-transmitted arboviruses most years; however, no prior studies have characterized the local epidemiological situation. Córdoba  is among the southernmost cities in the Western Hemisphere to report autochthonous dengue transmission, making it an important site to study the dynamics of the recent emergence of arboviruses in southern temperate latitudes. Córdoba also reported imported cases of chikungunya and Zika virus in 2014 and 2016, respectively, as well as at least one case of autochthonous Zika virus in 20166. Both chikungunya and Zika virus are transmitted primarily by the same vector as dengue virus.Within the last decade, dengue emerged for the first time in areas with temperate climates, including Córdoba, the second largest city in Argentina (population 1.3 million), located in the southern cone of South America 7. The Córdoba province Ministry of Health has reported no vector activity during winter months; therefore, it is hypothesized that local outbreaks originate from imported arbovirus cases, often in travelers from arbovirus endemic areas.Córdoba has a continental temperate climate, with warm summers  and dry, cool winters . Summer temperatures in Córdoba fall within the lower range of optimal temperatures for arbovirus transmission by Bacillus thuringiensis israelensis larvicide application, elimination of larval habitat)8. However, these efforts were unable to prevent outbreaks (as in 2016), and risk perception by the public remains low. Further investigation of the role of social and environmental drivers of arbovirus emergence in Córdoba is needed to develop effective vector control and disease management programs to reduce the burden of dengue illness.In response to the emergence of dengue fever, the Ministry of Health (MoH) of Argentina together with the Córdoba Entomological Research Centre from the National University of Córdoba and the Institute of Biological and Technological Research (IIBYT) have increased efforts to control vectors. Vector control by the MoH is mostly focal, around homes with dengue cases, and includes control of adult mosquito populations (indoor and outdoor fumigation) and larval mosquitoes  in Córdoba over the last decade. We obtained epidemiological data by manually extracting records from reports published by the Argentinian National MoH 6. The reports were published in Spanish and provided on a weekly basis in most years. We identified key data in the reports for characterizing arbovirus emergence, including the year-to-date cumulative number of probable  and laboratory confirmed autochthonous  and imported  cases of dengue, Zika, and chikungunya virus in Argentina. In some years the reports included information on serotypes of dengue cases and/or regions associated with imported cases . In Córdoba, epidemiological and vector surveillance are the responsibilities of the Epidemiology Area of the Zoonosis Program of the MoH of the province (https://www.cba.gov.ar/epidemiologiaweb/). Suspected dengue infections that are diagnosed at either private or public clinical sites are reported to the National Health Surveillance System operated by the MoH. A subset of clinically diagnosed cases are confirmed by laboratory diagnostics (polymerase chain reaction (PCR) and immunoglobulin M antibodies to DENV) at the province of Córdoba Central Laboratories . A subset of samples is sent to the Maiztegui Institute in Buenos Aires for confirmation using the plaque reduction neutralization test (PRNT). The same procedure is followed for all arbovirus cases (e.g. Zika and chikungunya cases).The data presented here were collected from public health reports provided regularly by the Argentinian National MoHWe aimed to collect all available data on arbovirus cases including probable and laboratory confirmed cases, autochthonous and imported cases, DENV serotypes, and origins of imported cases. However, this information was not available in all reports. We translated, compiled, and reviewed the data to determine the weekly number of cases, source of imported cases, and DENV serotypes in circulation. We consolidated all reported cases of dengue, Zika, and chikungunya for Córdoba city between January 2009 and December 2018 (135 weekly reports). We cleaned the data to correct inconsistencies in reporting: we eliminated cases that appeared to be counted multiple times, for example when a single case appeared to be reported as both probable and confirmed and sums of probable and confirmed cases did not equal sums of total cases. The cases presented in this database are a sum of probable and confirmed cases. In Córdoba, all unconfirmed cases presenting as caused by an arbovirus were assumed by the MoH to be a specific arbovirus (e.g. dengue) given their proximity to other confirmed cases. Between 2009 and 2018, approximately 94.2% of all dengue cases were laboratory confirmed. Additionally, 52.9% of chikungunya cases and 75% of Zika virus cases were laboratory confirmed.11 (https://www.indec.gob.ar/indec/web/Nivel3-Tema-2-41). The population size in each year, t, was estimated by the functionWe calculated the incidence of imported and autochthonous cases each week Fig. . IncidenNo custom code was created in the development of this database.12) as a set of three comma separated files with weekly time series data for dengue, chikungunya, and Zika cases and one comma separated file with summary data for dengue serotypes and origins of imported cases. Data for each file begins in the year that the virus was first detected in Córdoba . The column headings of the files with weekly time series data are as follows.The database is publicly available online IMP_CASES: The number of newly reported imported cases (cases reported with history of recent travel outside of the country)POP_SIZE: The population size of Córdoba in the YEAR of the entry (estimation method described in Methods)AUTO_INCIDENCE: The incidence (per 100000 people) of newly reported autochthonous cases.IMP_INCIDENCE: The incidence (per 100000 people) of newly reported imported cases.In the fourth file describing dengue case serotypes and origins of imported cases, the following column headings are used.YEAR: The year associated with the data presented.N: The total number of cases tested for serotypes (NA indicates the number tested was unknown or not reported).DENV: The total number of cases that were determined to be of serotype 1, 2, 3, or 4 as indicated by the column head.ORIGINS_GLOBAL: The known origins of imported cases that were contracted outside of Argentina.ORIGINS_ARGENTINA: The known origins of imported cases that were contracted in an Argentinian province other than Córdoba.In Fig. 4. The total dengue incidence in 2009 was 9.78 cases per 100,000 people.The first imported dengue case in Córdoba was reported Epidemiological Week (EW) 2 in 2009. In EW10, 2 cases of dengue in patients without travel to affected areas were confirmed, marking the first known autochthonous dengue case in Córdoba. In total, 88 autochthonous cases were reported between EW10-EW18. During EW2-EW18, 42 imported dengue cases were reported, leading to 130 total cases. All tested cases were confirmed to be DENV1In 2013, Córdoba experienced its second major outbreak, with 115 autochthonous and 10 imported cases reported . Autochthonous and imported cases were reported from EW7-18. The known origins of imported cases include Brazil, Bolivia, Mexico, Costa Rica, and Formosa Province in northeastern Argentina.In 2015, Córdoba experienced its third significant outbreak of dengue beginning with imported cases in EW5. From EW9-22, 221 autochthonous cases were reported. In total, 236 autochthonous and 14 imported dengue cases were reported . DENV1 and DENV4 were detected, and cases were imported from Brazil and Formosa Province, Argentina.Córdoba’s largest dengue outbreak to date began in EW52 of 2015 with an imported case of unknown origin. The first 2 autochthonous cases were reported in EW2, and 687 autochthonous and 134 imported cases were reported from EW2-24. In total, 822 cases of dengue were reported . Of these, 288 (35%) cases were tested for DENV serotypes  of dengue in Córdoba, which will be helpful to investigate potential impacts of vector control measures and eventual vaccine deployment in the city through mathematical modeling studies. Although complete serotype information on dengue cases is lacking in the present database, the information provided on the serotypes in circulation (i.e. from both autochthonous and imported cases) can be important for predicting outbreaks14. The overwhelming presence of DENV1 in outbreaks for which serotype information is available should give public health officials reason to take extra precautions in the event that transmission of other dengue serotypes occurs given the severe symptoms known to occur in secondary dengue infections1. Furthermore, the presentation of this data highlights that detailed clinical epidemiological data on dengue transmission is lacking and that future studies of arbovirus transmission in Córdoba should consider investigating serotype-specific seroprevalence across age groups (e.g via prospective serosurveys) and travel history of seropositive individuals.The data presented herein characterizes the emergence of dengue in the temperate city of Córdoba, Argentina, and when coupled with complimentary social-ecological datasets, will facilitate the further investigation of mechanisms that are driving this ongoing emergence, including potential links with climate, dengue serotype circulation, and global travel. Before 2009, Córdoba was considered to be a population entirely susceptible to dengue. The data from outbreaks of dengue occurring in 2009, 2013, 2015, and 2016 provide a unique opportunity to estimate the basic reproduction number , will be useful for estimating links between meteorological patterns and anomalies and dengue transmission, which could provide useful insights not only for Córdoba, but for temperate regions across the globe.In the last decade, 20. Regional efforts are needed to strengthen surveillance of arbovirus transmission, due to the key role of human movement in dengue introductions. The data presented in this work combined with arbovirus transmission data from other parts of Argentina as well as other countries in South and Central America, together with human movement data  could provide insights into the role that human movement has played in the ongoing emergence of dengue in Córdoba and provide guidance for preventing future outbreaks. The present study is focused on providing a detailed description of the arbovirus transmission data available for Córdoba in order to highlight in the recent and ongoing emergence of dengue in the city. Given the history of dengue importations from northern provinces of Argentina into Córdoba, an accessible nationwide database of arbovirus cases in Argentina would be critical for understanding patterns of transmission that lead to localized outbreaks in cities such as Córdoba . With the current political crisis in Venezuela, resulting in mass migration of people into Argentina, the risk of importation of dengue fever and other mosquito borne diseases  has increasedba Table .


In the article titled “Efficacy and Tolerability of Intravenous Ferric Carboxymaltose in Patients with Iron Deficiency at a Hospital Outpatient Clinic: A Retrospective Cohort Study of Real-World Clinical Practice” , there w(i) In the Statistical Analysis section, “The computed OR were adjusted for age and pretreatment hemoglobin for the endpoints related to hemoglobin increase and for age and pretreatment transferrin saturation for the endpoint related to the increase of transferrin saturation” should be corrected to “The computed OR were adjusted for age and pre-treatment hemoglobin for the endpoints related with hemoglobin increase, and for age and pre-treatment ferritin for the endpoint related with the increase of transferrin saturation.”(ii) There were multiple errors in the Efficacy Endpoints section. The corrected section is as follows.“The primary  and secondary  efficacy endpoints, following intravenous FCM treatment, are shown in Table 3. After 6 weeks, hemoglobin increase ≥2 g/dL was attained by 41% of all patients, 49% in the IDA group, 40% in the diseases of the digestive system group, 55% in the diseases of the genitourinary system group, 26% in the neoplasms group, and 29% in the diseases of the circulatory system group. Moreover, our analysis indicated that patients with IDA or diseases of the genitourinary system presented significant decreased odds of clinical failure , whereas patients with diseases of the circulatory system showed about three times higher odds of clinical failure . Regarding cumulative FCM treatment dose, we found significant decreased odds of clinical failure in patients who received doses ranging from 501 to 1000 mg  or 1001-3000 mg doses , compared to patients who received doses of 500 mg (Table 3).Hemoglobin increase of ≥3 g/dL after 6 weeks after FCM dose was attained by 20% of all patients, 25% of the patients in the IDA group, 22% in the diseases of the digestive system group, 26% in the diseases of the genitourinary system group, 11% in the neoplasms group, and 16% in the diseases of the circulatory system group. Furthermore, our analysis indicated that patients with IDA presented significant lower odds of clinical failure . Concerning total FCM treatment dose, our analysis showed significant lower odds of clinical failure in patients who received doses ranging from 501 to 1000 mg  or 1001-3000 mg doses , compared to patients who received doses of 500 mg (Table 3).Finally, transferrin saturation >20% after 6 weeks after FCM dose was attained by 63% of all patients, 62% of patients in the IDA group, 69% of patients in the iron deficiency without anemia group, 63% in the diseases of the digestive system group, 68% in the diseases of the genitourinary system group, 67% in the neoplasms group, and 47% in the diseases of the circulatory system group. Our analysis indicated significant lower odds of clinical failure in patients who received doses ranging from 501 to 1000 mg  or 1001-3000 mg doses , compared to patients who received doses of 500 mg (Table 3).”(iii) There were errors in Table 3. The corrected table is shown below.(iv) In the Discussion section, “For patients with IDA, no differences in treatment efficacy were found for hemoglobin increase of ≥2 g/dL and transferrin saturation > 20%, compared with patients without IDA. However, we found a significant difference in hemoglobin increase of ≥3 g/dL” should be corrected to “For patients with IDA, no differences in treatment efficacy were found for transferrin saturation >20%, compared with patients without IDA. However, we found significant differences in both hemoglobin-related efficacy endpoints.”Supplementary MaterialsThe underlying patient-level data and a script with all the logistic regression models adjusted to generate the Odds Ratio and respective confidence intervals for Table 3 are included as Supplementary Materials.Click here for additional data file.