Transvenous lead extraction (TLE) is the gold standard in the management of patients with cardiac implantable electronic devices (CIED)-related complications. Knowledge of TLE risk factors is very important. Clinical data from 1915 patients undergoing TLE at the Reference Center between 2006 and 2015 were analyzed. The effects of clinical and procedure-related factors on the development of major (MJC) and minor (MIC) complications and survival after TLE were evaluated. MJC were caused mainly by lead implant duration, presence of abandoned leads, multiple procedures preceding TLE, and any technical problem during TLE. Of clinical factors female gender and anemia increased the risk of MJC. MIC were reported in patients with the first implantation of CIED under the age of 30 and after sternotomy analysis of 30-day survival after procedure demonstrated a significant effect of clinical factors and lead dwell times, previous unsuccessful TLE, and MIC. Efficacy and safety of TLE depend mainly on procedure-related factors. This knowledge is essential for preventing MJC and MIC. Mortality at 30 days following TLE is mainly associated with the clinical factors; however, there was also a significant effect of lead dwell time and periprocedural complications on the short-term prognosis of patients undergoing TLE. Transvenous lead extraction (TLE) is the gold standard in the treatment of patients with CIED-related complications. Initial experiences in successful extraction of the leads by continuous traction were described in 1980 . Increased risk for developing of MIC (30.2 % by 1 g/dl) was related to lower hemoglobin concentration . The potential clinical factors significantly increasing the risk for developing MIC included female gender ; chronic renal failure (CRF) . The increased risk for MIC was additionally found in patients after sternotomy , implantation of mechanical or bioprosthetic valves , and with lead-related infective endocarditis (LRIE) , especially isolated LRIE , i.e., without signs of pocket infection (PI) .From factors that were directly procedure-related, the risk for developing MJC  and MIC was significantly increased in patients with older leads, measured as dwell time of the oldest one extracted lead in patient: MJC  and MIC , mean dwell time of leads extracted in patient: MJC  and MIC , and sum of dwell time of leads extracted in single patient: MJC  and MIC . The risk for developing MJC was also higher in patients undergoing atrial lead extraction  as well as simultaneous removal of leads from both sides of the chest . Extraction of abandoned leads (and their number) was associated with more frequent occurrence of MJC, respectively:  and . Removal of unipolar leads was associated with a significantly higher risk for developing MJC  and MIC . A significant increase in the risk of MJC was also observed in patients undergoing extraction of more than four leads , via the right-side approach  or requiring a more complex approach to lead extraction . Any technical problem during TLE also had a significant influence on the frequency of MJC , especially fibrotic adhesions binding the leads together which were associated with MJC occurrence  and periprocedural mortality   and MIC . The risk of complications increased significantly in relation with the number of abandoned leads ,  and TLE of leads implanted on both sides of the chest  .Potential device-related factors increasing the risk for developing MIC were sum of dwell times of leads in single patient before TLE , lead implantation under the age of 30 , and presence of intracardiac lead abrasions . TLE of defibrillation leads was connected with decreased risk of MIC by 60%   placed the patient at increased risk of periprocedural death and increased near five times the risk of death in 30-day follow-up   showed that the strongest values in prediction of MJC are age of the extracted leads (increase in risk by 3.1 to 18.1% per year), female gender (increase in risk from 2.36 to 2.91 times), number of abandonment leads in patients (increase in risk by 65.3 to 75.7%), number of any technical problems during TLE (increase in risk by 36.9%), and number of previous CIED procedures in patient (increase in risk by 27.6%). Lower by 1g/dl of blood hemoglobin concentration was associated with an increase in the risk of MJC occurrence by 22.4% to 27.4% .Risk of MIC was higher in patients with previous sternotomy , first implantation of CIED under the age of 30 , malposition of the lead in the left ventricle , lack of antiplatelet therapy , and lower blood hemoglobin concentration  .Multivariate Cox proportional hazards regression showed that the risk factors of death in 30-day follow-up were infective indications , a higher functional class of NYHA , chronic renal dysfunction , previous unsuccessful TLE , and MJC occurrence . Sum of dwell time of leads extracted in single patient also worsened the prognosis but these parameters reached the borderline statistical significance  .The Kaplan–Meier curves of time free from death depending on clinical success, NYHA class , infectious indications for TLE, and median of hemoglobin concentration in 30-day follow-up and results of log rang tests were presented in Transvenous lead extraction has become a common procedure worldwide. It is estimated that 10-15 thousand leads are removed annually and the need for lead extractions will probably continue to increase . The finIn the available literature, there is no detailed analysis of procedure-related factors. The world's largest registry of patients undergoing TLE at the Cleveland Clinic did not confirm the importance of procedure-related factors except the effect of lead implant duration on the development of major cardiovascular injury (MCVI) as well as the relationship between ICD lead removal and 30-day mortality in the univariate analysis. The multivariate analysis did not show the direct effect of the sum of lead dwell times, number, and type of extracted leads on the development of MJC . Small oThe presence of abandoned, superfluous leads was another extremely important predictor of adverse patient outcomes associated with TLE in the present study. There is still much debate about whether we should leave abandoned leads in place or extract them. Although the Cleveland registry did not demonstrate increased risk associated with the presence of abandoned leads, recent reports from the same center evaluating patients undergoing TLE for infectious reasons confirmed apart from more frequent occurrence of vegetations, a higher rate of complications developing during the procedure . AccordiOf the clinical factors in the present study female sex and low hemoglobin levels were the most important predictors of adverse outcomes following TLE. Although in previous investigations women were found to have more complications , 16, theIn the present study, apart from evaluating predictors of major complications, we analyzed also minor complications, which appeared to influence significantly 30-day mortality after TLE. Risk factors for MIC were similar to those observed for major ones; however, in the multivariate analysis, a significant effect of the implantation of the system at the young age and the history of sternotomy was demonstrated. A potentially higher procedural risk in patients with implanted devices younger than 30 years is especially worth emphasizing, because there is still a belief in the high risk of TLE in older people. It is however noteworthy that none of the available studies documented the effect of patient older age on the procedure efficacy. Moreover, previous investigators demonstrated the high efficacy of TLE in patients aged 80 and 90 years, comparable to that in younger subjects .The present study describes mainly procedure-related predictors of adverse outcomes in patients undergoing TLE; however evaluation of survival is very important. As shown in the present study 30-day mortality was affected by entirely different determinants, mainly the clinical ones such as heart failure, renal failure, and device infections. Most of these factors were demonstrated in the Cleveland registry . HoweverMultivariate analysis of such a large number of procedure-related factors is difficult to perform because of mutually exclusive determinants and marked predominance of one parameter over the other. In this situation, trivariate analysis was used to evaluate most reliably the effect of individual factors on the risk associated with TLE.Procedure-related variables have been found to be the most important predictors of adverse outcomes in patients undergoing transvenous lead extraction. A better understanding of these determinants allows for implementing strategies that minimize procedure-related risk and improve the efficacy and safety of TLE. Female sex and anemia were found to be the only significant clinical, patient-dependent factors. It is noteworthy that other clinical factors, frequently analyzed by other investigators such as heart failure, chronic renal failure, and pacemaker infections did not determine the immediate results of transvenous lead extraction. These factors together with minor complications influenced 30-day mortality after TLE and for this reason should be monitored closely in the periprocedural period.


Musculoskeletal and finite element modelling are often used to predict joint loading and bone strength within the human hand, but there is a lack of in vitro evidence of the force and strain experienced by hand bones.This study presents a novel experimental setup that allows the positioning of a cadaveric digit in a variety of postures with the measurement of force and strain experienced by the third metacarpal. The setup allows for the measurement of fingertip force as well. We tested this experimental setup using three cadaveric human third digits in which the flexor tendons were loaded in two tendon pathways: (1) parallel to the metacarpal bone shaft, with bowstringing; (2) a semi-physiological condition in which the tendons were positioned closer to the bone shaft.There is substantial variation in metacarpal net force, metacarpal strain and fingertip force between the two tendon pathways. The net force acting on the metacarpal bone is oriented palmarly in the parallel tendon condition, causing tension along the dorsum of the metacarpal shaft, while the force increases and is oriented dorsally in the semi-physiological condition, causing compression of the dorsal metacarpal shaft. Fingertip force is also greater in the semi-physiological condition, implying a more efficient grip function. Inter-individual variation is observed in the radioulnar orientation of the force experienced by the metacarpal bone, the fingertip force, and the strain patterns on the metacarpal shaft.This study demonstrates a new method for measuring force and strain experienced by the metacarpal, and fingertip force in cadaveric digits that can, in turn, inform computation models. Inter-individual variation in loads experienced by the third digit suggest that there are differences in joint contact and/or internal bone structure across individuals that are important to consider in clinical and evolutionary contexts. Different manipulative and locomotor behaviours lead to varied biomechanical loading of the hand and affect the external morphology (e.g. entheses) and internal structure  of bones. The ability of bone tissue to remodel in response to the magnitude and direction of load is known traditionally as Wolff’s law  or, now With recent advances in 3D imaging techniques, the internal bone structure of human, nonhuman primate, and fossil human (hominin) hand bones has been investigated to help reconstruct hand function and behaviour in extinct taxa. Variation in cortical and trabecular bone structure has been linked to the differences in hand loading during locomotor behaviours in living apes vs. manipulation in humans . It has Understanding the loads experienced by the hands is also important within a clinical context, including research on joint disease , trauma Because both in vivo and in vitro studies of hand bone loading present several technical challenges, musculoskeletal models are typically used to predict force during isometric hand functions, such as pinch and power grip tasks . The staThe purpose of this study is to help to fill this gap of information on in vitro hand bone loading. Here, we present a novel experimental setup that allows simultaneous measurement of in vitro loading and deformation of human and nonhuman primate hand bones during functional postures. The experimental setup also allows the measurement of fingertip force. This experimental design builds upon previous in vitro studies that typically only measure fingertip force in cadaveric specimens . We testTwo clamps were designed to fixate a cadaveric digit at its fingertip and its metacarpal base. The concave cavities of the clamps were designed to hold the fingertip and the metacarpal base of the specimen . A compact data acquisition system  and a customized program  were designed to acquire the metacarpal force and strain signals simultaneously. To measure the fingertip force, the load cell was attached to the distal clamp and the distal metal stand. The same data acquisition system was used to acquire the fingertip force data.Although the experimental setup can be used for each digit, we tested the setup on the third ray as this digit plays an important role in the daily activities of humans  and nonh2  joint at 25° flexion, the proximal interphalangeal (PIP) joint at 60° flexion, and the MCP joint at 55° flexion as this joint configuration is at the approximate midpoint of the functional range of motion required to perform 90% of daily manipulative activities . In addi2  and a PCectively . This spUnlike previous studies that only measured the fingertip force in cadaveric specimens , we needFollowing the quantification of the force and strain experienced by the metacarpal, the strain gauge wires were removed and the load cell was fixed to the distal clamp and the distal stand for the direct measurement of fingertip force. The same posture, loading conditions and tendon pathways were applied as described above.First, the force and strain experienced by the metacarpal bone were collected synchronously at 100 Hz, and then just fingertip force data was acquired at 100 Hz. The acquired force and strain signals were calibrated with baseline data collected when the flexor tendons were not loaded. Then, the signals were filtered using a sixth-order Butterworth low-pass filter with the cut-off frequency at 6 Hz. For the parallel condition, the average force and strain values within the middle 3 seconds of data collection were calculated. For the semi-physiological condition, a single frame of data was extracted since the tendon path was dynamically modified. All analyses were conducted within a customized MATLAB script  that were applied to the specimens to avoid rupture of the tendon, thus resulting in small force and strain measurements. Accuracy error of the sensors and the noise in the signals may bias the results. However, the accuracy error of the load cell was less than 0.05 N see . Also, tThis study used the same tendon load for three specimens while there might be individual difference in the anthropometry of the soft tissues. Due to the logistical challenges of measuring the PCSA of each specimen prior to the experiment, this study used the published mean PCSA values of the human FDS and FDP  to estimFinally, the parallel and semi-physiological tendon pathways used in this study may not be ideal for simulating the tendon path within a hand with intact soft tissues. The parallel tendon pathway is a simplified condition that, although not fully representative of the biological condition, is particularly valuable for building informative musculoskeletal or FE models and obtaining replicable predictions. Although the artificial positioning of the tendons and the data extraction method could be improved, this study provides the best possible approximation of the normal physiological state within the current experimental constraints.Although this study presents the force and strain measured in human third digit only, the experimental setup is adjustable to fit variation in the size and morphology of other human and nonhuman primate digits, as well as varying digit postures. Future studies could also include loading of more tendons and/or different loading patterns. Comparison among different digits with varied functional postures and tendon loading patterns may inform research on osteoarthritis, prosthetic design, and the interpretation of internal bone structure within the human hand. Comparative studies between humans and nonhuman primate digits can help inform the interpretation of the differences in external and internal bone morphology in relation to locomotor and manipulative behaviours. This comparative context is needed for more robust functional interpretations of fossil hominin hand morphology and the reconstruction of hand use throughout human evolution.This study presents a novel experimental design for simultaneous in vitro measurement of metacarpal force and strain. In addition, the experimental design allows for the quantification of fingertip force. Testing this experimental setup on three human cadaveric fingers revealed substantial difference in force and strain between two tendon pathway conditions, which, in turn, emphasizes the importance of tendon path simulation in computational models. The in vitro data presented in this study are not only useful for musculoskeletal and FE modelling in hand-related research such as prosthesis design, but also for the functional interpretation of the variation in internal bone structure within humans. In the future, investigation of forces and strain in different fingers could contribute to a better understanding of the varied prevalence of the MCP joint osteoarthritis across the digits . In the 10.7717/peerj.5480/supp-1Supplemental Information 1mc) is presented in the proximal(+)/distal(−), dorsal(+)/volar(−), and radial(+)/ulnar(−) directions with respect to the metacarpal bone. The strain gauge (SG) measurement was performed at three sides of the bone, i.e. radial-palmar, dorsal, and ulnar-palmar sides. The fingertip force (Ftip) is presented in the proximal(+)/distal(−), dorsal(+)/volar(−), and radial(+)/ulnar(−) directions with respect to the distal phalanx.This table presents the force and strain data measured from the three specimens in two tendon path conditions, i.e. parallel and semi-physiological conditions. The force experienced by the third metacarpal bone (FClick here for additional data file.10.7717/peerj.5480/supp-2Supplemental Information 2The load cell was tested before being used in the experiment. The error was less than 0.05 N and the full-scale accuracy error was less than 0.1%.Click here for additional data file.10.7717/peerj.5480/supp-3Supplemental Information 3The precision of strain gauge measurement was verified via beam models, and the analytical calculations showed the same strain pattern and similar values as the experimental results. Photo credit: Dr Szu-Ching Lu.Click here for additional data file.10.7717/peerj.5480/supp-4Supplemental Information 4The acquired force and strain signals were calibrated with baseline data collected when the flexor tendons were not loaded. Then, the signals were filtered using a sixth-order Butterworth low-pass filter with the cut-off frequency at 6 Hz.Click here for additional data file.


Drosophila tissues that undergo similar shape changes. We will also put forward a hypothesis for optic cup folding that considers an active contribution from the RPE.The neural component of the zebrafish eye derives from a small group of cells known as the eye/retinal field. These cells, positioned in the anterior neural plate, rearrange extensively and generate the optic vesicles (OVs). Each vesicle subsequently folds over itself to form the double-layered optic cup, from which the mature eye derives. During this transition, cells of the OV are progressively specified toward three different fates: the retinal pigment epithelium (RPE), the neural retina, and the optic stalk. Recent studies have shown that folding of the zebrafish OV into a cup is in part driven by basal constriction of the cells of the future neural retina. During folding, however, RPE cells undergo an even more dramatic shape conversion that seems to entail the acquisition of unique properties. How these changes occur and whether they contribute to optic cup formation is still poorly understood. Here we will review present knowledge on RPE morphogenesis and discuss potential mechanisms that may explain such transformation using examples taken from embryonic  All the organisms of the animal kingdom that bear a visual sensing organ share the need of protecting the light-receptive cells with a pigmented structure . In the The RPE is a neuroectodermal derivative. In zebrafish, its specification begins in cells that occupy the dorso-medial portion of the optic vesicle (OV), which is the first morphologically recognizable primordium of the eye . All OV The adult zebrafish eye shares strong similarities with that of other vertebrates but its initial morphogenesis occurs with slightly different mechanisms. In amniotes, eye progenitors are bilaterally positioned in the anterior neural plate  and protDrosophila embryo there are examples of tissues undergoing a similar conversion. The underlying mechanisms have been studied and could provide clues regarding RPE flattening, as we detail below.As the ventral/lateral layer expands and undergoes shape changes, the remaining cells of the dorsal/medial OV layer begin to acquire RPE identity and transform their appearance Figure . SimilarDrosophila imaginal disks are among such examples. These epithelial sac-like structures present in the larva originate most of the adult appendages during metamorphosis  as well as a YAP-related control of cytoskeletal dynamics (Figure opo mutants (With these considerations in mind, folding of the zebrafish OV into a cup could be modeled as a morphogenetic process that requires the contribution of the dorso/medial and ventro/lateral layers. The expansion of the apical retinal layer may be an initial trigger that exerts tension on the overlying layer, thereby forcing YAP nuclearization and the activation of Tead-mediated transcription of tissue determinants (i.e., s Figure . Changess Figure . These cs Figure . The rim mutants , in whic mutants . In cont mutants .otx morphants and Otx null mice, in which the RPE does not form, the neural retina is everted (Other studies support an active role of the RPE in OC morphogenesis. The RPE can bend autonomously in culture . In eye- everted , indicatThus, it seems reasonable to propose that tension and extension of the RPE can be crucial for proper OC folding just as much as basal constriction of the retinal cells or epithelial flow at the rim. This hypothesis needs experimental verification.otx genes have a similarly essential role in RPE specification but mitf genes seem to be dispensable (yap inactivation gives a phenotype that differs from that of the mouse, as already discussed. These differences might be explained by the zebrafish genome duplication or the presence of additional related genes that have adopted their function as proposed in the case of mitf (4D analysis of the embryonic zebrafish has provided a wealth of information on eye morphogenesis, although full comprehension of RPE formation is lagging behind and many questions remain open. For example, there are still uncertainties on how many of the movements sustaining zebrafish eye morphogenesis can be fully applied to the formation of the amniote eye. Meanwhile, details of the gene regulatory network leading to zebrafish RPE specification are unclear. In mice, this network includes Wnt-βcatenin signaling and the activity of the transcription factors Yap, Otx, Mitf followed by Pax6 and Sox9, which both contribute to maintain RPE identity . In zebrpensable , whereas of mitf . AlternaTM-M, FC, and PB conceived and wrote the manuscript.The 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.


The Jinjiang horse is a unique Chinese indigenous horse breed distributed in the southern coastal areas, but the ancestry of Jinjiang horses is not well understood. Here, we used Equine SNP70 Bead Array technology to genotype 301 horses representing 10 Chinese indigenous horse breeds, and we integrated the published genotyped data of 352 individuals from 14 foreign horse breeds to study the relationships between Jinjiang horses and horse breeds from around the world. Principal component analysis (PCA), linkage disequilibrium (LD), runs of homozygosity (ROH) analysis, and ancestry estimating methods were conducted to study the population relationships and the ancestral sources and genetic structure of Jinjiang horses. The results showed that there is no close relationship between foreign horse breeds and Jinjiang horses, and Jinjiang horses shared a similar genetic background with Baise horses. TreeMix analysis revealed that there was gene flow from Chakouyi horses to Jinjiang horses. The ancestry analysis showed that Baise horses and Chakouyi horses are the most closely related ancestors of Jinjiang horses. In conclusion, our results showed that Jinjiang horses have a native origin and that Baise horses and Chakouyi horses were key ancestral sources of Jinjiang horses. The study also suggested that ancient trade activities and the migration of human beings had important effects on indigenous horse breeds in China. There are over five million horses in China, the majority of which are indigenous horses that are distributed widely across the rural areas of China. The largest horse populations exist in the northern and southwestern provinces . The JinHowever, the origin of Jinjiang horses remains unknown due to a lack of historical records. There no longer exist indigenous horse breeds in the provinces adjacent to Fujian Province, namely, the Zhejiang Province, Jiangxi Province, and Guangdong Province, which make the ancestry of Jinjiang horses more elusive. Because Quanzhou, where Jinjiang horses are distributed, was the most important and pivotal port of the Marine Silk Road (AD 12 to AD 14) . A studyThus far, most phylogenetic studies on horse populations have been based on mitochondrial data ,10,11,12In the present study, the genome-wide high-density SNP arrays were employed to genotype horses from 10 Chinese indigenous breeds representing the five horse breed groups of China, which were classified according to the geographic distributions and genetic relations of the indigenous breeds, namely, the Mongolia Group, the Hequ Group, the Tibet Group, the Kazakh Group, and the Southwest Group. Additionally, the genotyped genomic SNPs data of 14 foreign horse breeds from previous studies  were alsBlood samples of 301 horses from 10 CHBs and five donkeys were collected. The samples were obtained following the principles approved by the Animal Care and Use Committee of China Agricultural University (Permit Number: XK257). Genomic DNA was isolated by standard phenol/chloroform extraction methods.The sampled horse breeds included the Mongolian horse (IMG) from Inner Mongolia, representing Mongolia breed group, the Daan horse (DA) in Jilin Province (belonging to the Mongolia horse group), the Chakouyi horse (CKY) in Gansu Province (representing the Hequ breed group), the Naqu horse (NQ) in Tibet (standing in for the Tibet breed group), the Kazakh horse (KZK) in Xinjiang (representing the Kazakh breed group), and horse breeds from South China  in Guangxi Province, the Tengchong horse (TC), Zhaotong horse (ZT), and Lijiang horse (LJ) in Yunnan Province, and the Jinjiang horse (JJ) in Fujian Province, which was the only breed from Southeast China ). Five dp-value < 1 × 10−5; (2) SNPs that were missing more than 10% of their genotype data; and (3) SNPs with a minor allele frequency less than 1%. Individuals with more than 10% missing genotyped data were also removed. The total SNPs left in the merged dataset of the worldwide horse populations were 39,187, while there were 57,597 filtered SNPs in the thinned data of the 10 Chinese indigenous breeds  distance matrix between individuals was generated by PLINK v1.07  using thTo study the relationship between the Jinjiang horse and the other CHBs, the 57,597 filtered SNPs were applied to conduct the subsequent analyses. A phylogenetic tree merely containing the Chinese horses was constructed with the same steps mentioned above. An analysis of population structure was carried out using the software Admixture v1.3 , which uR2) between any two loci (within and between different chromosomes) using PLINK v1.07 [Linkage disequilibrium (LD) levels for Chinese horse populations were assessed by the genotype correlation coefficient  detection. The frequencies of shared haplotypes between Jinjiang horses and each of the other CHBs were estimated with per 10,000 bp bins using IBDLD software (v3.37) . The parROH), number of ROH(NROH), and autozygosity (FROH) were computed.Runs of homozygosity segments (ROH) were determined with an overlapping window approach implemented in PLINK v1.07 . The finO) and expected heterozygosity (HE), as well as the inbreeding coefficient (f), were also determined with PLINK v1.7 using the command --het.Indices of genetic diversity, including observed heterozygosity  was calculated with the model covariance matrix (W), as a function of modeling the number of modeled migration events. More migration edges did not seem to further increase the variance explained by the phylogenetic model, as the f index reached an asymptote above 6 migration edges D, inset,Because the Jinjiang horse is solely genetically related to other CHBs according to the results of the analyses of the worldwide horse data, the subsequent studies were focused on the 10 CHBs to investigate the genetic relationships between the Jinjiang horse and other Chinese horse breeds in the following analyses using the 57,597 SNPs that passed quality filtration. A neighbor joining (NJ) tree only containing Chinese indigenous horses showed that Jinjiang horses form four branches, with most Jinjiang horses following their geographical distributions and clustering with horse breeds from Southwest China, while a few individuals of the breed are relevant to horse breeds in North China A. Artificial selection tends to reduce genetic diversity, and commercial breeds have undergone stronger artificial selection than local breeds have. The impact of artificial selection could also be detected in the genome LD levels in each population, as artificial selection can facilitate the increase of LD within a population . The resThe results from both analysis of ADMIXTURE K = 4, C and PCAROH can quantify more recent inbreeding. In the horse populations studied, the highest FROH was observed in the Andalusian horses (mean FROH = 0.1022), followed by the Thoroughbreds (mean FROH = 0.0990). The lowest value was obtained for the Chakouyi horses (mean FROH = 0.0060), and the Naqu population also had very low FROH values. FThe observed heterozygosity of the horse breeds ranged from 0.2955 to 0.2748 . The MorROH, most of the individuals in the population possess the ROHs, and the inbreeding coefficient (f) of the breed indicated that Jinjiang horses currently may have the highest inbreeding level among the Chinese horse breeds studied.The Jinjiang horse population showed medium values of the ROH parameters compared with other Chinese indigenous horse breeds. Although only a moderate degree of inbreeding occurred in the Jinjiang horse population in the past, based on the estimated value of FROH and FROH. The results are consistent to the breeding history of these horse breeds.Compared with the Chinese indigenous horse breeds, the foreign breeds, which have been subjected to highly selective breeding, such as Thoroughbreds, Andalusian horses, Arabian horses, Morgans, Shetland ponies, and Akhal_Teke horses, have higher NThe results of IBD analysis showed that Jinjiang horses are closely related to horse breeds in Southwest China . The Baihttps://david.ncifcrf.gov/) is provided in Long consecutive homozygous genotype segments, i.e. ROHs, result when parents transmit identical haplotypes, which can be used for estimating autozygosity. Thus, we used PLINK software to compute the number of ROHs. Through investigating the distribution of ROHs across the genome, we detected the highest number of ROHs on ECA 11 of Jinjiang horses . An overAlthough above studies suggest that the Jinjiang horse has a diverse native-Chinese origin, the possible ancestry of Jinjiang Horses still remains unknown. Therefore, the f4 Ratio Estimation algorithm /f4 ) from ADMIXTOOLS Software Package  were useALDER v1.03 software  was applThe earliest records about horse raising in Jinjiang of Fujian Province was in the Tang dynasty (AD 618-AD 907), which was much later than that of North China, where the remains of the earliest domestic horses in China were found. It was also later than the most horse-raising areas of Southwest China. It is unclear from where the early horses in Jinjiang were introduced. Because Quanzhou city was the most important port in the Marine Silk Road and there were frequent and prosperous trading activities between Quanzhou and the Arab world , it was To study the relationship of the Jinjiang horses and the worldwide horse populations, we created a merged dataset of 580 Chinese and foreign horses from 10 CHBs and 14 foreign horse breeds with 39,187 autosome SNPs, which were used to construct phylogenetic NJ trees (five donkeys used as outgroup). It was indicated that the Jinjiang horses have close relationship with other CHBs but did not show direct genetic link to any foreign horse breeds. Admixture analysis results also showed that the Jinjiang horse has the similar ancestral background to other CHBs, which is obviously different from foreign horse breeds. PCA and TreeMix analyses further confirmed the results of the clustering analysis and showed the close genetic relationship between Jinjiang horses and the other Chinese indigenous breeds, and there was no gene flow between Jinjiang horse and foreign horse breeds. These results showed that there is no significant genetic association between Jinjiang horses and foreign horses, and the Jinjiang horse was not influenced by the foreign breeds in history. Some of our results also showed that there is a close genetic relationship between European heavy horses and northern Chinese horses, especially with Daan horses, which was also confirmed in the TreeMix analysis. These phylogenetic results are in accordance with the breeding history of Daan horses, which had been bred by hybridizing the mares from local breeds with the sires of European heavy horses . The genn = 234 and 57,597 SNPs) was involved in the subsequent analyses. The phylogenetic NJ tree (plus five donkeys as an outgroup) showed that most of the Jinjiang horses have a close relationship with the horse breeds in Southwest China, while a few individuals of the breed are relevant to the northern horses, which is attributed to the low selection imposed on the indigenous breed. Some stallions, which have no significant blood relationship, were used in breeding activities and were the founders of the subpopulations. These results are also in accord with the output of the LD analysis. The lowest LD level of Jinjiang horses suggested that Jinjiang horses did not experience intensive selection and that the genetic components of their ancestral sources may be largely preserved. The PCA and Admixture analysis confirmed the results of the phylogenetic NJ tree about the close genetic links between Jinjiang horses and the southwestern horse populations. The results of estimating genome-wide IBD sharing indicated that Jinjiang horses are most closely related to Baise horses among the southwest Chinese populations. Our study also revealed an evident admixture of Chakouyi horses and Jinjiang horses, suggesting a complicated history of gene flow from Chakouyi horses to Jinjiang horses. Further analysis revealed that Baise horses and Chakouyi horses are two of the most important ancestors of Jinjiang horses, and they account for approximately 47% and 20% of the ancestral sources of Jinjiang horses, respectively. Thus, the ancestry of Jinjiang horses is the mixture of Southwest and North Chinese horses. It is far away from the distributing areas of Baise horses (Baise of Guangxi Province) or Chakouyi horses (Tianzhu of Gansu Province) to those of Jinjiang horses (Jinjiang of Fijian Province)—i.e., over thousands of kilometers. The gene flows from Baise horse and Chakouyi horse were inevitably related with the historical trades and migrations of human beings, which may give reasonable explanation about the long-distance migrations of the horses. Because Jinjiang horses are only closely related to Chinese indigenous horses, we focused on the Chinese indigenous horses. Thus, the dataset of the 10 Chinese horse breeds (The ancient Tea-Horse Road and Marine Silk Road in China may have a significant impact on the formation of the Jinjiang horse. From the Tang Dynasty (AD 618—AD 907) to the Qing Dynasty (AD 1636—AD 1912), the ancient Tea-Horse Road traversed through Yunnan province and Sichuan province to Tibet, which primarily aimed to trade tea from the tea-producing areas for horses from Tibet and other regions of horse-raising, but eventually it developed into arteries for trade in the mountainous regions of Southwest China, not only limited to Tea-Horse trading activities. In the mountainous ancient Tea-Horse Road, packhorses were used to cross difficult terrain, and horse-packing was the main means of transport for goods, while the businessmen who managed the horse-packing trade were called horse caravans. Guangxi Province, where high-quality tea is also produced, is at the eastern end of the ancient Tea-Horse Road. The ancient Heng-shan-zhai city of Guangxi Province, adjacent to Baise, had been an important and prosperous trade center in the Song Dynasty (AD 960–AD 1297). The ancient trade and the Tea-Horse Road facilitated the migrations and exchanges of horses in these areas . The resThe Baise horse is an important horse breed in Guangxi Province. Because Guangxi Province was a major transportation and trade area , which connected inland provinces (such as Yunnan Province and Guizhou Province) to the coastal provinces, including Guangdong and Fujian where important ancient ports were located, the Baise horse had a profound influence on other horse breeds in the whole southwest region . The MarChakouyi horses are mainly distributed in the Gansu Province. The province is at the northern end of the Tibet-Yi Corridor, which represented the long routes (over 1000 km) along the north-south oriented rivers and valleys in the boundary of the southwest and northwest provinces of China. By following the corridor, people could migrate from the northwestern provinces to Southwest China, including Yunnan Province, Sichuan Province, and Tibet, all of which are areas along the ancient Tea-Horse Road . AncientThe hypothesized routes by which Baise horses and Chakouyi horses migrated to Fujian Province are shown below . TherefoAfter the population of Jinjiang horses continuously decreased in the past several decades, Jinjiang horses have become an endangered horse breed with a small population size and the conservation of the breed is now a pressing matter to carry out. From this study, the population structure and the genetic relationship of Jinjiang horses were elucidated, and the high level of inbreeding was also revealed with the analysis of ROH and the genetic diversity. The subpopulations of the breed could be used as main lines for further breeding programs and conservation. Moreover, the results regarding the genetic relationship among the individuals of the breeds also provide useful information for establishing sound mating systems for the population, thereby avoiding further inbreeding depressions. The ancestry sources revealed in the study can also be introduced to rejuvenate the Jinjiang horse. Although obvious association between the function of genes in the selected segments and the specific traits of Jinjiang horses was not found, the results provide valuable information for further studies to illustrate the signatures of selection, which offer meaningful information for the conservation of the breed. The results of our study suggest that Jinjiang horses originated from other Chinese indigenous breeds. The Baise horse and Chakouyi horse are two of the main ancestral sources of the Jinjiang horse. Ancient trading activities and the migration of human beings had deep impacts on the formation of the coastal horse breed.The Jinjiang horse has been bred from their ancestors and is adapted to coastal southeast China, and it is essential to take action to conserve the indigenous breed and develop a new breeding strategy to improve the equestrian traits of the breed, through which it can eventually be developed into a high-quality saddle horse breed.


These pathological conditions affect endogenous neurogenesis in two aspects. On the one hand, injury usually initiates the migration of neuronal precursors (NPCs) to the lesion area from the already existing, in physiological conditions, neurogenic niche – the ventricular-subventricular zone (V-SVZ) near the lateral ventricles. On the other hand, recent studies have convincingly demonstrated the local generation of new neurons near lesion areas in different brain locations. The striatum, cortex, and hippocampal CA1 region are considered to be locations of such new neurogenic zones in the damaged brain. This review focuses on the relative contribution of two types of NPCs of different origin, resident population in new neurogenic zones and cells migrating from the lateral ventricles, to post-stroke or post-traumatic enhancement of neurogenesis. The migratory pathways of NPCs have also been considered. In addition, the review highlights the advantages and limitations of different methodological approaches to the definition of NPC location and tracking of new neurons. In general, we suggest that despite the considerable number of studies, we still lack a comprehensive understanding of neurogenesis in the damaged brain. We believe that the advancement of methods for  At present, it is well known that the production of new neurons in the mammalian brain is not restricted to the embryonic and early postnatal development stages, but occurs throughout the lifespan of animals. However, not all brain regions are equally capable of generating new neurons. There are two main neurogenic niches where neurogenesis persists: the ventricular-subventricular zone (V-SVZ) and the subgranular zone of the hippocampus (SGZ). By the term V-SVZ we mean a complex of brain regions that includes: the subventricular zone (SVZ) – the area stretching along the lateral wall of the lateral ventricles, the ventral extension of the lateral ventricles, and the posterior periventricular region (PPV). Several researchers have also considered these areas as a single neurogenic zone . During Some types of brain damage, such as ischemia or traumatic brain injury (TBI), stimulate neurogenesis in the brain . On the Here we consider approaches to studying neurogenesis, paying particular attention to how the origin of new neurons can be established. In other words, whether new neurons have originated in the same area where they localize, or if they/their precursors have migrated there from another area. Methods for defining migration pathways of NPCs are also reviewed here.Historically, the first studies that established the production of new neurons in the mammalian brain were performed using autoradiographic investigation of 3H-thymidine-labeled dividing cells, in particular, newborn neurons . HoweverThis approach, based on the administration of such proliferation markers as BrdU and other analogs of thymidine, still remains among the most widely used methods. BrdU can be administered intraperitoneally or with drinking water. It incorporates into the cells that are in S-phase at the time of administration. The timing of BrdU administration and subsequent killing of animals affect the interpretation of results. When BrdU is given systematically for several days or weeks, labeled cells born at different time points cannot be distinguished. Alternatively, a single dose (pulse) of BrdU labels the cells that are born at the time of the pulse. Then animals can be sacrificed either shortly after administration, or at a delayed time point . This approach allows for the tracing of a short-term or a long-term fate of a particular group of cells that are born at a certain time point .In addition to BrdU, other nucleotide analogs can be used: -5-chloro-20-deoxyuridine (CldU), 5-iodo-20-deoxyuridine (IdU), and 5-ethynyl-20-deoxyuridine (EdU). The use of combination of several thymidine analogs within the same experiment gives additional possibilities. Labeling of dividing cells with two  or even The immunodetection of BrdU or other thymidine analogs in fixed brain slices, in combination with labeling the specific markers of immature (doublecortin or DCX) and mature  neurons, as well as NSCs , allows for indirect tracking of NPCs. Additionally, Ki67, a marker of cell divisions, is used to confirm the proliferation status and to identify the location of non-migrating progenitors .Although this approach facilitated the estimation of the proliferation and differentiation of NPCs, it provided only indirect evidence of neuroblast migration. The signs of migration can be: a distribution pattern of migrating neuroblasts (markers – DCX and sometimes PSA-NCAM) from the place of origin toward the place of destination , a densi14C-dating was proposed by Jonas Frisen’s group , and striatum -3,3,3′,3′ tetramethylindocarbocyanine (DiI), and fluorescent latex microspheres are used. For detection of the labeled cells, fluorescent microscopy is used . Howeverin vivo cells tracing encode many different reporter genes that allow for the identification of targeted cells. These include the green fluorescent protein (GFP) gene, the yellow fluorescent protein (YFP) gene, the DSRed, mCherry, and mKate for detection using fluorescence microscopy; the alkaline phosphatase (AP) gene for immunodetection; the galactosidase (GLA) gene for histochemical detection using transmitted light microscopy  in the construct results in non-specific expression of the reporter gene in the cells that have been successfully infected with a genetic vector . NeverthInterestingly, there is still at least one work in which this approach was applied to investigate the origin of new neurons in the brain. A completely different approach involves the use of transgenic animals already carrying a reporter gene in their cells. As a rule, the reporter gene is initially inactive because of an insertion containing a stop signal. It can then be restored (the restoration mechanisms are described below), thus facilitating the reconstruction of the reporter gene in the cells residing in a specific brain region at a specific time point and of a specific phenotype. Later, the cells where the reporter gene was restored and their offspring can be detected .The reporter gene can be restored by the use of site-specific Cre-recombinase that removes an insertion from the reporter gene . A speciLastly, Another principally new technique to have been recently developed is the Brainbow strategy  which aiin vivo cell labeling is the most reliable method for establishing the origin of new neurons in brain regions. Rapid development in genetic engineering led to widespread using of plasmid libraries designed for new viral vector production. To increase efficiency of the use of libraries, a novel tool of viral barcoding was suggested , and brain lipid-binding protein (BLBP) . HoweverIn the early postnatal brain, several migratory pathways stretching from the V-SVZ to other brain regions have been described. In the adult brain, the V-SVZ remains the main zone of neurogenesis. However, its intensity decreases soon after birth and the majority of pathways become largely quiescent . The excThe migration from the V-SVZ to other brain regions substantially decreases compared with the early postnatal period, and a range of works have confirmed this. Despite the fact that these pathways remain largely quiescent , they mi14C technique showed that in the OB, adult neurogenesis can be negligible. As in animal models, neurogenesis in the SVZ in humans declines over a lifetime (In humans, the amount of neuroblasts migrating to the OB drops rapidly in the early postnatal period. lifetime .The DG of the hippocampus ranks second after the V-SVZ where proliferation of NPCs continues throughout the whole life of animals. According to the classical point of view, the dentate neuroepithelium gives rise to both granular neurons and NSCs in the prenatal period . HoweverUnlike the SVZ , neurogeAsymmetric division gives rise to bipolar neural progenitors that differentiate into neuroblasts after several divisions. During their maturation these NPCs, unlike NPCs from the V-SVZ, migrate short distances – from the subgranular to the granular layer of the DG – and become mature glutamatergic granular neurons .In adult humans, hippocampal neurogenesis in the DG is more prominent than in the OB . SpaldinNeurogenic potential of the cerebral cortex in physiological conditions is considerably smaller in comparison to the V-SVZ and to the SGZ of the DG.The majority of cortical neurons and glial cells originate from the radial glia stem cells that located along lateral ventricles. These cells generate cortical neurons and direct their migration to the cortical layers at the embryonic stage . This prAlmost no neurogenesis occurs in the cortex of the normal adult brain in rodents, but systematic analysis shows that small numbers of NeuN/BrdU double positive new neurons still continue to be generated there . Their ode novo generated neurons have transient existence because their number declines after 9 weeks of observation. The origin of these cells has not been studied in this work. In humans, Eriksson’s studies (14C incorporation, estimated the exchange rate of neurons in the adult human neocortex at 1/1,000 neurons every fifth year (A number of studies in primates and humans mainly report on the absence or a low level of adult neurogenesis in the cerebral cortex. Longitudinal observations by  studies  showed afth year .For several decades, the piriform cortex has attracted significant attention due to its ability to express the markers of immature neurons DCX and PSA-NCAM, which was revealed in most mammalian species in adulthood. Nevertheless, numerous attempts to achieve evidence of neurogenesis in this area using BrdU labeling failed. It was concluded that the expression of DCX and PSA-NCAM could be explained by this particular population of neurons retaining its structural plasticity .The adult striatum, at least in some mammalian species, also retains the potential to generate new neurons. Ontogenically, the striatum originates from the ganglionic eminence located in the ventral part of the developing telencephalon. The most active striatal neurogenesis, which proceeds in two phases that result in a different functionality of striatal neurons, almost ends with birth . In adulUnlike the cerebral cortex, there is significantly more evidence of the generation of new neurons in the adult striatum under physiological conditions. Several works did not reveal new neurons in the unlesioned striatum . NeverthThe origin of NPCs in the adult striatum has been intensively studied. Some works have largely clarified the question concerning “local vs. SVZ” source of these neurons. Various pathologic changes and injuries strongly affect neurogenesis and migration of NPCs in the adult brain. Though the V-SVZ is still considered the main source of new neurons, migration pathways and destinations of NPCs can change depending on lesion area. At the same time, there is much evidence that new neurons can originate from different sources outside the known and proven neurogenic zones. In the next section we consider several works investigating these changes.In this section we describe changes in proliferation and migration of NPCs in the injured brain. In general, injury can stimulate V-SVZ neurogenesis accompanied by the migration of V-SVZ-derived neuroblasts to the site of injury, as well as local generation of new neuroblasts near the damage area. In the latter case, new neuronal progenitors are found to arise from local astrocytes, which gain NSCs properties. The significance of SVZ-derived versus local progenitors is a highly debated question. As has already been mentioned, under physiological conditions significant neurogenesis occurs in the DG of the hippocampus. Under ischemic conditions, mainly in the model of global ischemia, the number of neuroblasts in the DG can grow , 2018a.Additionally, after global ischemia new neurons are detected in the CA1 region of the hippocampus, where in these conditions a massive death of pyramidal neurons occurs . The PPVThus, these data testify that new neurons in the damaged CA1 region originate from the PPV. However, there is no proof that resident NPCs cannot occur in this region after an injury, because this possibility has not been specifically examined.In the damaged striatum, many young neurons can be detected, as has been shown on models of ischemia  and neonSome works do not specify the origins of these cells . However-labeling cells in the SVZ ;-direct observation of migration in living brain slices ;-intraventricular administration of the antimitotic agent Ara-C ;-or estimation of neuroblasts distribution in fixed brain slices .Additionally, data provided by At the same time, three works proved that after lesion, a number of new neurons in the striatum are generated from local precursors . Nato etIt should be noted here that the majority of the above-mentioned works estimated the role of either the SVZ or only of the striatum in the production of new neurons in the damaged striatum. Only the two works investigated both and obtained contradictory results . In partDamage to the cortex of the adult animal brain enhances neurogenesis in it, and this has been shown in different models of brain damage, such as ischemia , neonataThe majority of works establish their origin from the SVZ or from the PPV. These findings were obtained with the use of different approaches based on:-distribution pattern of neuroblasts migrating from the SVZ/PPV to the damaged area in fixed brain slices ;-suppression of cell proliferation in the SVZ by intraventricular administration of Ara-C ;-labeling cells from the lateral ventricles ;-and direct observation of migration in the live brain .At the same time, some studies do in fact suggest that the production of new neurons can occur in the injured cortex . Bi et aSome works described that after permanent MCAO in adult animals, cortical astrocytes , 2012, aDespite a large number of studies, it is still difficult to get a full picture of neurogenesis in the damaged cortex since the majority of researchers estimated the role of either only the cortex or of the SVZ in the production of new cortical neurons. The work by In this section we consider several works that describe interhemispheric migration of neuroblasts after cortical injury . The autAlthough this review focuses mainly on the anatomical origin of new neurons appearing in the damaged brain areas, the question about their functional relevance cannot be ignored due to its high importance when seeking out new directions for future therapies. Here we discuss some related issues. For more information about the survival, differentiation and functional integration of new neurons within the injured brain tissue, we direct the reader to the reviews by Relatively few works have addressed the study of structural and functional integration of new neurons into the existing neural networks after brain injury. Mainly, such integration has been shown in the damaged striatum and cortex.At least two studies provided evidence that both SVZ-derived and locally generated neurons may successfully integrate into the pre-existing circuit within the damaged cortex. Several studies showed structural and functional integration of new neurons into the existing striatal networks. Despite these optimistic results, the majority of these studies showed two important limitations in the restoration of neuronal circuits by adding newly generated neurons: the low survival rate of these neurons and the poor scope of their differentiation potential. Most new neurons within the ischemic striatum die, possible due to unfavorable environment in the damaged tissue . Since gin vitro. It seems unlikely that these strictly predetermined cells can produce other types of neurons necessary for brain recovery.Another important limitation is a relevance of functional features of integrated new neurons to the pre-existing functionality of the network. Studies on a phenotype of newly generated neurons within the ischemic striatum provided contradictory results regarding a percentage of newly generated neurons expressing DARPP-32, a marker of medium-sized spiny neurons – the major class of striatal neurons. Identification of the causes of the above-mentioned discrepancies is one of the directions for future studies. Thus, more research needs to be done to comprehensively evaluate the regenerative potential of adult neurogenesis and to develop optimal strategies for its enhancement.Up to the present time, a substantial amount of research material concerning the origins and migratory pathways of NPCs in the brain under physiological and pathological conditions has been accumulated. In the healthy brain, the majority of NPCs originate in the V-SVZ located in the walls of the lateral ventricles . In the 14C-content assessment (Human neurogenesis, which has been also shown in the studies with the comprehensive sessment , substanDespite the impressive array of research works, at present there is no full picture of neurogenesis and NPC migration in the brain. Most research works address either a single neurogenic region or a single migratory pathway of neuroblasts. Future studies on the injured brain could examine the following aspects: firstly, the local or migratory nature of NPCs after brain injury could be confirmed by novel techniques of real-time observations; secondly, since the presence of newly generated neurons does not ensure their functionality, the question about the integration of new neurons into the existing networks and their phenotypes could be further investigated; thirdly, comparative studies are needed to understand the difference between human and rodent neurogenesis for future translation of new therapies to clinic.in vivo visualization of neurogenesis in the brain could fundamentally change the current situation in this field.It is likely that in the future, methods for longitudinal observation capable of capturing new neuron production, migration, and functional integration in the same animal brain, will be improved. We believe that the advancement of methods for NN-D wrote the manuscript. MK conceived the presented idea and critically revised the manuscript. Both authors contributed to the literature search and analyses.The 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.


Many reconstruction methods are performed for combined defects of upper lip and premaxilla in oral cancer patients, which are complicated and multiple staged procedures, compromising the functional or structural unit. In this case report, we present a modification of the bilateral perialar crescent flap for reconstructing the combined defect of upper lip and premaxilla in a single stage. A patient diagnosed with well-differentiated squamous cell carcinoma of premaxilla and upper lip, involving a surgical defect of more than one-third but less than two-thirds of the lip underwent two cycles of neoadjuvant chemotherapy. Later wide local excision of the lesion and simultaneous reconstruction with a modified perialar crescent flap was performed in a single stage. Patient recovered uneventfully and tolerated the procedure well, without any complications. The patient was found to be satisfied with the functional and esthetic result. The reduced upper lip support which was a result of the bony defect of the premaxilla, was corrected with a dental prosthesis. This modification is a useful reconstruction tool for oral cancer patients with combined defects of upper lip and premaxilla. Reconstruction of the upper lip and pre-maxillary segment is one of the greatest challenges for a surgeon. They both play a vital role in facial esthetics and oral function. The lip and its complex subunits make the reconstruction a very artistic and skillful procedure. If the tumor involves the pre-maxilla segment, upper gingivobuccal sulcus and is in direct continuity with the upper lip, then resection in the form of partial maxillectomy with extensive loss of upper lip tissue becomes mandatory. Full-thickness defects of one-third of the upper lip can be simply restored by direct apposition of the wound edges. When the defect is extensive and requires reconstruction of the lateral and medial small topographic subunits like philtrum, alar base, and nasolabial fold, a local or distant flap may be required . CrescenA 60-year-old female presented to the oral oncology outpatient department with painful ulceration in the upper lip, involving the gingivobuccal sulcus and premaxilla region of five months’ duration. Patient’s history revealed that she started using prosthesis for missing upper anterior teeth, after which the swelling started insidiously and progressed rapidly to attain the present size.Clinical examination revealed a diffuse swelling in the upper lip, approximately 3 x 3 cm in maximum dimension, involving the alar base and philtrum. Intraoral examination revealed an ulcero-proliferative lesion crossing the midline; involving the anterior maxillary alveolus and gingivobuccal sulcus from 13 to 23 and traversing more than one-third of the upper lip .To reduce the size of the lesion and decrease the possibility of positive margins during resection, a medical oncologist’s opinion was sought, who suggested neo-adjuvant chemotherapy (NACT) with a combination of docetaxel, cisplatin, and 5-fluorouracil. Reassessment after three weeks following the first cycle of NACT showed a marked response with a considerable reduction in the size of the lesion. The second cycle of NACT was administered and on reassessment, the lesion had further reduced considerably Figure .The surgical plan was wide local excision of two-thirds of upper lip with anterior maxillectomy and reconstruction with bilateral modified perialar crescent flap for the resulting surgical defect. This treatment plan was proposed considering the site and size of the lesion, and the feasibility to achieve adequate closure while reconstructing with the desired flap. Under general anesthesia, resection with 1 cm surgical margin for the lip lesion and 1.5 cm for premaxilla lesion was planned and markings made with indelible ink. Full thickness resection of the upper lip lesion and anterior maxillectomy including the first premolars bilaterally was performed. The excised specimen was sent for histopathological examination Figure .The medial and lateral vertical incision of the Burow’s triangle was placed along the nasolabial fold and the plane of dissection was kept below the underlying muscle. The crescentic flap margins for excision were kept wider and lower in this case, and the medial margin was extended around the alar base. The triangular portion of the Burow’s flap was rotated down intra-orally bilaterally, covering the surgical defect of the premaxilla region. Both the pedicled flaps were approximated cautiously and sutured together with the adjacent tissue margins. Buccal mucosa was freed and undermining of the cheek was done, therefore resulting in a full thickness cheek flap. Following this maneuver, the remnants of the lip were advanced to close the defect. The lip was sutured in layers and a temporary obturator was given for labial fullness Figure . Due to Oral competence and speech were good and the patient was able to perform a wide range of facial expressions. One month postoperatively wound healing was satisfactory without oro-nasal communication or neurosensory complications Figure .There are various surgical modalities for correction of the combined defects of upper lip and premaxilla region which involve multi-staged procedure thereby compromising function and aesthetics. For combined defects of the premaxilla and upper lip, no single-stage procedure has been documented, as it requires both ablative and reconstructive surgical procedures for restoration of the defect. In our case, a modified bilateral perialar crescent flap was utilized for reconstructing the combined defect of premaxilla and full-thickness defect of the upper lip to achieve satisfactory results in a single stage.In stage IV lesions, multimodality treatment should be favored, depending upon the site and extension of lesion . Based oFor the reconstruction of the upper lip defects, the reconstruction ladder starts from the simplest procedure and ascends in its complexity with emphasis on esthetics and conserving lip function. The aesthetic subunits of the upper lip reconstruction were elegantly described by Burget and Menick and are derived from the pertinent topography . In the In our case, after wide local excision of the tumor, there was loss of premaxilla and portion of the upper lip. In the repair of full-thickness defects of the upper lip, bilateral perialar crescent flap enables better structural alignment of lip commissures and nasal unit with good functional results; but the loss of the premaxillary segment cannot be corrected in the same procedure . Hence, There were no complications associated with wound healing, adaptation, and vascularity of the flap. The advantages of this technique include reconstruction of combined upper lip and premaxilla in a single-stage thereby deferring the need for complicated secondary reconstructive procedures and preventing morbidity to the patient.The limitation with the modified bilateral perialar crescentic advancement flap is microstomia . The narModification of the bilateral perialar crescentic flap is a reliable technique that offers satisfactory functional and esthetic results. It is useful in reconstructing combined defects of upper lip and premaxilla in a one-stage surgical procedure, which reduces surgical cost and donor site morbidity.


Recently, droplet-based microfluidic systems have been widely used in various biochemical and molecular biological assays. Since this platform technique allows manipulation of large amounts of data and also provides absolute accuracy in comparison to conventional bioanalytical approaches, over the last decade a range of basic biochemical and molecular biological operations have been transferred to drop-based microfluidic formats. In this review, we introduce recent advances and examples of droplet-based microfluidic techniques that have been applied in biochemistry and molecular biology research including genomics, proteomics and cellomics. Their advantages and weaknesses in various applications are also comprehensively discussed here. The purpose of this review is to provide a new point of view and current status in droplet-based microfluidics to biochemists and molecular biologists. We hope that this review will accelerate communications between researchers who are working in droplet-based microfluidics, biochemistry and molecular biology. In the past three decades, biochemistry and molecular biology have been developed in various basic research areas and applications in which microfluidics have played one of the most common roles ,2. It haIn recent years, droplet-based microfluidic (DMF) technologies have been focused on reducing fluids to the nano-to-femtoliter scale. This reduction makes the flow in the channels become laminar, represented by a low-Reynolds number, which gives derivate properties like the immiscibility of the different phases of the fluid. It changes the fluid’s properties as the flow in the channels will be laminar, represented by a low-Reynolds number, which gives derivate properties like the immiscibility of the different phases of the fluid. Under the aforementioned conditions the samples are divided into droplets for better manipulation of the properties of the assays . To prevGenes, proteins and cells are fundamental building blocks of life and have been tremendously studied in biochemistry and molecular biology to understand the mechanism of life. In this review, we briefly introduce selected recent advances of DMF technologies for biochemistry and molecular biology, with particular emphasis on genomics, proteomics and cellomics, then highlighting also the possibilities in biological applications.Complete genomic studies of organisms in all other respects, including whole genome sequencing, directed evolution, quantification of DNA/RNA, and even epistatic, pleiotropic and heteroidal interactions of genes, are the most important in understanding the function of genes in biochemistry and molecular biology fields. In recent years, DMF technologies have been merged with molecular biology approaches for the understanding and also for engineering genetic information through whole genome sequencing and quantification of nucleic acids . This spAmong the different techniques used in genomics, DNA sequencing is probably one of the most important tools for understanding genetic information. DMF technologies have advantages in terms of throughput, sample amount and assay time for the identification of DNA and also for the single cell-based study. Single-molecule droplet barcoding (SMDB) is one of excellent examples of an ultrahigh-throughput method to barcode long DNA molecules for short-read sequencing . Using DPCR (polymerase chain reaction) technology has been evolved with the use of DMF technologies for the precise detection and absolute quantification of the nucleic acid of interest b. BasicaCampylobacter jejuni) through hybridization with a DNA mixture containing pathogenic DNA. Then, target DNA was amplified with fluorescently labeled target-specific primer within picoliter droplets and Luminex bead were collected for the analysis in the Luminex instrument.Zhao and co-workers recently introduced oil-saturated polydimethylsiloxane (PDMS) chip (OSP) for the on-chip droplet digital PCR (ddPCR) that was used for absolute quantification of lung cancer related microRNA and pathogenic bacterial DNA ,24. EastNucleic acid amplification is a key procedure for the detection and quantification of biomarker nucleic acids. Conventional PCR amplification requires repeated cycles of three or two temperature-dependent steps during the amplification processes which requires a thermocycler. In contrast, isothermal amplification methods have no need for thermal cycling that can be used in simplified-microfluidic systems including DMF-based ddPCR. Various isothermal amplification methods have been developed and merged in DMF for precise detection and absolute quantification of the nucleic acid of interest .Plasmodium) in blood and saliva by integration of DMF with RCA [Plasmodium topoisomerase I (pTopI) in the saliva of the patient [Rolling circle amplification (RCA) consists of an isothermal enzymatic process where a long single stranded DNA or RNA is formed using a circular template for the amplification of a short primer a. The prwith RCA . Leong a patient . Multiple displacement amplification (MDA) allows massive parallel amplification of single cell genomes while maintaining the accuracy and specificity of the sequence b. TakeyaLoop-mediated isothermal amplification (LAMP) uses multiple primer sets for a single nucleic acid target. It consists of an auto-cycling strand displacement DNA synthesis that is catalyzed by DNA polymerase with high strand displacement c 48]. R. R48]. RContinuous-flow digital LAMP is another innovation for the integration of this amplification technique with droplet-microfluidics, which has been already utilized for ultrasensitive DNA detection . The groHelicase dependent amplification (HAD) consist of using a probe with a hairpin structure that bears the transcription factor binding site to convert the protein signal to the DNA signal. Cao and colleagues demonstrated a simple method for the detection of a transcription factor. In the absence of a transcription factor zero background signal can be achieved due to the digestion of excess probes by the exonucleases and the subsequent one primer-triggered high fidelity amplification . Recombinase-polymerase amplification (RPA) uses two oligonucleotide primers specific to the target, which binds to the template DNA assisted by a recombinase in combination with strand-displacement DNA synthesis d. RecentUnderstanding proteome, structure and function of proteins in tissues is the next generation challenge in molecular biology. In particular, throughput is one of the important factors in the study of protein expression and its structure, and this is due to the vast amount of data from various tissues and cells . RecentlThe structure of proteins has been studied in molecular biology and biochemistry for a long time and even nowadays it is one of the most common topics in this field. Protein crystallization at a droplet scale allows the use of reagents and protein samples to be minimized which contributes to reducing the cost of the process and at the same time to controlling the diffusion of molecules and the nucleation of crystals ,64,65. MProtein quantification methods increased their efficiency and sensitivity using DMF as it has been demonstrated with the development of digital amyloid quantitative assay (d-AQuA). The d-AQuA uses DMFs for absolute quantification of single insulin paragons at a single molecule sensitivity with a phase of amplification by serial dilutions. This approach is being possible to be used for the monitoring of protein misfolding and aggregation (PMA) diseases . QuantitProtein–protein interaction studies, immunoassays and enzymatic assays are the main examples of how DMF technologies have been utilized to determine the functions of proteins. Enzymes are challenging to engineer due to the complexity of relationship between the sequence and biochemical properties. DMF technology can provide ultrahigh-throughput methods for mapping the enzyme sequence–function relationship by combining droplet microfluidic screening with next-generation DNA sequencing .−1 of C-reactive proteins ..6 varian8 reactions/day compared to the 73,000 reactions per day that can be analyzed with previous technologies . W. W94]. W2O2 that provides −2e−  is used, followed by hybridization with fluorescent probes. The number of transcripts in every cell is assessed by simply counting fluorescent dots inside gel beads . DMF tecE. coli was lysed with lysozyme and the intracellular target cleaved the DNAzyme sensor which triggers fluorescence. Then, fluorescent droplets are quantified by using the 3D fluorescent particle counter, allowing the single-droplet detection in a few milliliters of unfluorescent droplets  is one of the most popular technologies for single cell based biological experiment because it can be used to sort single cells according to their size and fluorescence intensity. However, some issues are raised due to their size, cost and complexity for users which makes it difficult to use frequently. Recently DMF technology has provided an alternative approach for single cell sorting which can be potentially used as a cheaper and easier method. The analysis and sorting of single mammalian cells is one of the most common procedures in molecular biology research. Cells can be manipulated and the sorting might be divided into active when an external force like magnetic, acoustic or electrical are used for the sorting  or passiL. lactis strain MG1363, JC017, which secreted low levels of riboflavin. Using fluorescence-activated droplet sorting, it has been shown that various mutants that secreted riboflavin more efficiently than JC017 were readily isolated from the mutagenesis library, then candidates with as few as 1.6 mutations per million base pairs (Mbp) were isolated with a highly efficient screening . . 133]. FLast years, modification and even synthesis of complete cells has been a major tool for research in which reducing the scale allowed better approaches and results. DMF technology has been proved as a very effective tool for the development of new cell engineering techniques. Among them CRISPR is probably one of the most exciting topics in molecular biology that has emerged these last few years; combined with droplet microfluidics tools, the delivery of CRISPR has been used for gene therapy to knock down the TP53BP1 gene from K562 cells ,136. DMFA very interesting application of DMF technology is the construction of synthetic cells. The assembly of unilamellar vesicles and compartmentalization by pico-injection, merging lipid vesicles and copolymers-stabilized droplets for generating stable cell-like compartments, have been called droplet-stabilized giant unilamellar vesicles (dsGUVs) . A doublDMF technology can produce monodisperse droplets controling the size precisely and tailoring the internal structure to incorporate different cells in designed locations. For example, Chen and colleagues reported a new method for the development of organ-on-a-chip for the first time producing highly monodisperse structures that enable multiple types of cells to arrange them to create an artificial liver in a drop .Recently, biochemist and molecular biologist have particular interest in accomplishing the challenge of providing useful technologies which can be used in real life problems such as biomedical diagnosis and among other fields like drug screening and development. Probably one of the most used tools in these diagnostic field is PCR. DMF technologies update this procedure like we have already discussed in this paper. Digital PCR can provide a robustness and convenience which can mitigate the effects of PCR inhibitors in diluted samples, regardless of the form of template  for forensic short tandem repeat (STR) typing at the single-cell/molecule level .Among all the possible applications of DMF technology, clinical diagnosis is one of the most promising fields which can benefit from this technology by increasing the throughput and reducing the time needed to obtain the results which is of paramount importance when we turn to diagnosis.The detection of RNA and DNA levels in DMF devices is a powerful tool for diagnosis of various diseases like infections or cancer; several examples have been shown such as controlling the levels of overexpression of genes in various cell lines .Sensing biomarkers is another application that has improved these past few years, showing various examples like detecting the levels of glucose which can be sensed more precisely with DMF electrochemical sensors using platinum-black electrode generated by micro deposition of Pt nanoparticles on bare Pt microelectrode. The levels of glucose oxidase can be detected up to 43.5 mM offering possibilities for developing droplet-based highly sensitive and low consumption sensors . MultiplIn the past decade, DMF technology has been integrated in devices already commercialized like the Bio-Rad digital droplet PCR system which has been used for multiplex DNA targets quantification . RaindanDrug screening is one of the applications which can benefit greatly from DMF technologies allowing the performance of high-throughput screenings in a shorter time. There are various DMF-based approaches have been developed and widely applied for drug screening. Recently for the screening of more than 56 conditions and 20 replicates on different pancreatic cell lines, regulating the flow of the different 16 syringes to produce different combinations . In theiRecent advances in DMF technologies for biochemistry and molecular biology have been introduced and discussed in this review. It can be seen that biochemistry and molecular biology have found in DMF a technology for improving the sensitivity, accuracy, speed and throughput of experiments . The advances in sequencing and detection, and protein and single cell detection in DMF can be a great boost for basic research and clinical purposes. However, more advances in the development of devices for point of care testing has to be achieved, integrating DMF devices in complete instruments will be one of the challenges for the next few years in the field  Table 2Table 2.


The U.S. is facing a shortage of aging-related professionals. Lack of positive attitudes among undergraduate students towards the older population may restrict them from participating as aging professionals. However, research majorly focused on the attitudes among students already in the medical training fields instead of improvement of the attitudes among undergraduate students who potentially will devote themselves to be aging professionals. We seek to enhance attitudes toward the aging population through gerontological coursework at the undergraduate level. The introductory course is based on Cognitive Behavioral theory (CBT) which emphasizes to unlearn false concepts. Therefore, the class exposes students to positive aging images in contemporary films and literature demonstrating concepts such as successful aging, retirement, gender issues, and aging as minorities, etc. Students review their perceptions of the aging population at the beginning and end of the semester, presenting five words that best describe the aging images. The variances of occurrence of negative to positive descriptions are analyzed as an evaluation of the class. The class also stimulates civic responsibilities of the students toward the aging population with concepts such as generational equities by conducting team-based discussions. The results report increased positive words and improved attitudes on in-class team projects and post-class evaluations. The implication of the course outcomes is that discussing and exposing the positive images, and justifying typical aging-related behaviors significantly improves students’ attitudes toward the aging population, and may encourage undergraduate students to select an aging profession. Qualitative evaluation of the course will be conducted in future semesters.


Microcrack formation and delamination growth are the main damage mechanisms in the fatigue of composites. They lead to significant stiffness loss, introduce stress concentrations and can be the origin of subsequent damage events like buckling or fibre breakage, especially in case of shear and compression stresses during load reversal. Fatigue experiments of carbon fibre reinforced laminates were conducted at several stress ratios and analysed in terms of crack and delamination growth. These investigations were accompanied by microscopic imaging, digital image correlation and finite element modelling to take into account the effects of residual stresses and crack closure. It was found that residual stresses significantly change the local stress ratio in off-axis layers and lead to residual crack opening of inter fibre cracks. These cracks remain open and close under high compression loadings only. Furthermore, crack formation under pulsating compression loading turned out to be driven by residual stresses leading to perpendicular cracks as observed under pure tension loading. The experimental findings further confirm the severe detrimental effect of tension-compression loading on crack formation and delamination growth compared to pulsating tension-tension or compression-compression loads. High performance composite structures usually undergo complex loadings throughout their service lifetime. Whereas failure of composite materials under static loading conditions is fairly well understood and many damage models exist ,5,6,7,8,Particularly in fatigue loading, many different factors influence the damaging process, e.g., the type of fatigue load , environmental conditions , material configuration and manufacturing processes ,17. FurtComposite microcracking is in focus of research for many years. Early works from Reifsnider et al. ,24,25 asGamstedt et al.  discusseQuaresimin et al.  used glaSimilar findings for carbon fibre reinforced (CFRP) cross-ply laminates tested at different load ratios published by the authors in earlier work  support The microcracks within the embedded off-axis layer are often origins for the initiation and growth of inter-layer delaminations. Delaminations can grow fast with catastrophic effect on laminate stiffness, strength and stability, because there are no crack stopping mechanisms between the two adjacent layers. Reviews by Bak et al. , Khan etKardomateas and Malik ,20 analyThis short literature review indicates that most research focusses on one damage mechanism or loading in tension or compression seperately. There is still a lack of experimental evidence and explanations of the detrimental effect of load reversals on the fatigue damage behaviour of composite laminates. Therefore, the aim of the presented study is to provide experimental results, further insight into the mechanisms which govern the fatigue failure of composite laminates and to derive explanations for the observed phenomena with special attention to the influence of tension-compression loading. Further attention has to be paid to residual stresses affecting composite materials due to their highly anisotropic material properties, especially in case of CFRP cross-ply laminates ,32. The Different specimen types were manufactured to determine the elastic and strength properties of the single layer as well as failure behaviour of cross-ply laminates. The single layers consist of T700SC 6k carbon fibre rovings from Toray International Inc. and Araldite LY556 epoxy resin system from Huntsman LLC.In a first step, the unidirectional (UD) as well as the bidirectional (BD) lay-up is built by a dry winding process on to a plate-type winding core with rounded edges providing two laminate stacks at a time with a layup of The specimens used for material characterisation were cut by water-jet to their specific dimensions according to the corresponding standards ,35,36,37The elastic properties of the composite material are determined by testing of unidirectional specimens in terms of tensile, compression and shear  experiments. Strain gauge rosettes are applied to both sides of the specimens loaded in tension and compression to monitor possible bending and to determine the Poisson’s ratios. Most of the specimens exhibit bending strains below 5%. If the tested specimens show higher bending strains throughout the test, the testing results are rejected and not considered in the calculation of the mean and standard deviation of the material properties. The VNRS experiments are accompanied by digital image correlation (DIC) for strain measurement. Therefore it is possible to observe the strain field within the complete notched area. Subsequently, the shear modulus is calculated from the stresses and strains between the two notches. The elastic material properties are given in It should be noted, that the standard deviations of the fibre parallel compressive strength i according toThe quasi-static analysis of the cross-ply laminates is performed by means of a Zwick Z250 testing machine with a 250 kN load cell at standard atmosphere  performed by the help of a Vallen Systeme AMSY-5 measurement system with two wideband sensors of the type VS150-M with a frequency range from 100 The cyclic experiments are conducted on a servo-hydraulic Instron 8801 universal testing machine equipped with a 100 kN load cell and mechanical wedge grips. All tests are performed at constant stress amplitudes and load control. To avoid buckling and premature failure due to geometrical stability issues, the specimens are supported by an anti-buckling device clamped onto the specimen. Polytetrafluorethylen (PTFE) films are placed between the contact areas of the specimen and the anti-buckling device to minimise friction and avoid abrasion. The laminate strain is measured by use of an extensometer attached to one of the free specimen edges. A linear variable differential transformer (LVDT) is used to monitor possible bending throughout the cyclic experiments and to stop the test in case of undesired buckling. Additional utilisation of a climate chamber avoids unpreferred specimen heating and reduces the influence of environmental effects. A photograph of the test setup as well as a schematic of the anti-buckling device are given in Four different load ratios according to The test schedule is summarised in o. 4 see , no initThe results of the crack density analysis together with the corresponding AE measurements and representative stress-strain curves of cross-ply laminates tested up to ultimate failure are shown in ergy cf. b. There loadings ,39. HoweIn To further analyse the influence of microcracks on the laminate behaviour it is of interest whether the cracks close under reversed loading or remain open. The crack opening displacement (COD) of a crack is investigated since it has been found to be an important parameter and can be used to calculate the residual laminate stiffness as well as the energy release rate for microcracking ,42,43,44As pointed out in the literature the amount of residual stresses in a laminate is important to be considered in an appropriate damage analysis ,45. In aA two-dimensional finite element (FE)-model was set-up in Abaqus CAE to determine the amount of residual stresses leading to the measured CODs. The FE-model consists of fully integrated plane-strain elements (CPE4), periodic boundary conditions at the specimen edges in loading direction and assumes linear elastic material behaviour without delaminations at the crack tips. The elastic properties from Considering load reversals within fatigue tests it is of particular interest to investigate the opening and closing of cracks. Therefore one specimen was studied in detail throughout one load cycle by DIC at the specimen edge and the strains in the cross-ply laminate were examined layerwise cf. a.First, the specimen has been statically loaded in tension to introduce a significant amount of cracks and subsequently loaded in tension and compression of different magnitudes. Additional to the DIC measurements, the global laminate strains were recorded by an extensometer at the opposing specimen edge. It was therefore possible to analyse the strains between two adjacent cracks experimentally. As seen from Assuming linear elastic material behaviour and perfect bonding between the 0equation (2)u˜CODThe calculated CODs along the unloading path for pure tension and a tension-compression cycle are given in Regarding Considering an averaged COD of The analysis of the fatigue behaviour focusses on the determination of the active damage mechanisms. Hence, the crack development has been observed regarding the aspects: amount of cracks, crack growth rates and cracking angle inclined to the loading direction. The results of the crack counting are given in In general the microcracking process is more pronounced in case of reversed loading . Therefore, the remaining part of the Regarding the images in  R=−3.26 c appearsFrom the results shown in Consequently, the macroscopically applied load ratios are shifted into the direction of pulsating tension loading, meaning that the macroscopically fully reversed experiments (The second issue of interest is the initiation and growth of delaminations. The fatigue growth behaviour varies between pulsating and reversed loading, as it can be seen from As stated before, two specimens failed prior to A reason for the differences in delamination growth between specimens tested with and without load reversals might be the change of fracture modes that are active throughout the cyclic loading. The delamination in tension loading can mainly grow under mode II, whereas delamination growth under compression loading has a significant mode I contribution at the tip of the delamination as observed by . This miIn contrast to these findings based on residual crack opening, Wevers et al.  proposedIt should be pointed out, that the results obtained here are acquired from a comparatively small set of eight specimens. The crack density evolution in The study presents experimental results for the microcracking process in CFRP-laminates in static and cyclic loading. It further provides data for the fatigue growth of inter-layer delaminations emanating from the tips of inter fibre microcracks and the remaining crack opening displacement due to manufacturing induced residual stresses in a cross-ply laminate.The results show the significant influence of residual stresses to fatigue loading conditions by shifting the local load ratios within the By use of microscopic imaging, the residual crack opening displacement was measured and used to calculate the residual stresses within the From edge investigations the delamination growth was measured for each load ratio. It was found, that delamination growth is more pronounced in case of reversed loadings (