Klinefelter Syndrome (KS) is characterized by an extreme heterogeneity in its clinical and genetic presentation. The relationship between clinical phenotype and genetic background has been partially disclosed; nevertheless, physicians are aware that several aspects concerning this issue are far to be fully understood. By improving our knowledge on the role of some genetic aspects as well as on the KS, patients’ interindividual differences in terms of health status will result in a better management of this chromosomal disease. The aim of this review is to provide an update on both genetic and clinical phenotype and their interrelationships. In 1942, Klinefelter et al. . KS has KS is associated with several clinical conditions coming from both the genetic abnormalities and hypogonadism. The aim of this review is to discuss KS clinical features according with the genetic and hormonal (low testosterone) factors involved in their pathogenesis.The major signs and symptoms of Klinefelter Syndrome (KS) have been well characterized since the first description of the disease . As tradClinical features depend on both the supernumerary X chromosome and the effects of hypogonadism . HoweverIn spite of the clinical phenotype of men with KS, as classically described in the literature, a parallel, less described phenotype has also been recognized, in which patients present with fewer clinical features . These lThe high frequency of mild phenotypes explains, at least in part, why most of the patients with KS remain undiagnosed  and claiSigns and symptoms appearance depend also on patients’ age. Furthermore, the phenotype tends to worsen with advancing age Fig. , accordiThe main sign, which is always present in KS, is represented by small testes. At puberty, both the sexual development and the growth spurt generally proceed in a normal way, but the progressive increase in testes volume does not occur, both testes remaining small (<4 mL in volume) and firm , 10, 11.The degree of virilization varies widely in adult men with KS, but it shows a tendency to decrease and to worsen progressively with advancing age Table , similarThe genetic background for the KS is based on sex chromosome non-disjunction, which leads to the presence of extra X chromosome/s. Indeed, non-disjunction represent the failure of chromosome to separate at anaphase during meiosis I, meiosis II or mitosis giving rise to cells with an aberrant number of chromosomes. This could happen either during oogenesis or spermatogenesis  or, less frequently (about 3 %), during early division of the fertilized egg.The occurrence of the maternal or paternal meiotic non-disjunction appears equally distributed in the KS patients (nearly 50 % each) . In KS pThe origin of the supernumerary X chromosome has also been associated with phenotypic differences, although evidence is not conclusive. In particular, it has been reported that KS patients with a paternal origin of the supernumerary X chromosome have a later onset and slower pubertal progression . Other sAn advanced maternal, and possibly paternal, age has been reported as a risk factor for KS. A 4-fold increase in the prevalence of KS cases was showed in mothers aged >40 years, compared to mothers aged <24 . The matMosaicism  is present in around 10–20 % of the KS patients and arises from either non-disjunction in an early mitotic division of the developing 46,XY zygote, or from loss of one of the X chromosome of a 47,XXY conception due to anaphase lagging.Y KS mouse animal model is comparable with the one observed in female subjects [Y KS mouse model. They are equally or less expressed in the liver and kidney of, respectively, 40,XX or 40,XY mice, while they are more expressed in the brain of the 41,XXY mice compared to the normal karyotyped mice, either male or female.In the somatic cells of females, the transcription of one of the two X chromosome is known to be randomly inactivated in order to ensure a dosage-compensation of the X-encoded genes to that of male cells. Although several genes are escaping inactivation, the Barr body (sex chromatin) in female cells is microscopically identifiable and represents the visible inactivated X chromosome . The untsubjects –37. Thessubjects  and the subjects , 40 probsubjects –46, and subjects . The gensubjects  have demMoreover, the skewed X chromosome inactivation, defined as the preferential inactivation of one of the two X chromosome in female, is present in the KS patients as well and this phenomenon may influence the clinical phenotype.The situation in the germ cells seems to be different and more complex since the X inactivation in these cells follows a distinct pathway , 49. EarThe androgen receptor (AR) gene, which mapped to Xq11.2-12, is of physiological importance in the testis and may play a particular role in differences of the KS phenotype. The N-terminal domain of AR gene exon 1 contains a stretch of CAG repeats, which is highly polymorphic. The length of this stretch is inversely correlated with the receptor activity . In KS pHowever, other studies , 63 did The pseudoautosomal regions (PAR1 and PAR2) are short homologous regions between the X and Y chromosomes in mammals. The PAR behave like an autosome and recombine during meiosis. Thus genes in this region are inherited in an autosomal rather than a strictly sex-linked manner . PAR1 isOther than gene dosage effects and parental origin of the supernumerary X chromosome, recent evidence suggested that additional features of the X chromosome might have a role in phenotypic differences among KS subjects. In particular, it has been found that KS subjects have more frequently than controls X-linked copy number variations (CNVs)  . The numHypogonadism remains silent until pubertal onset. Data on serum testosterone and estradiol in healthy prepubertal children are scanty, and there are no studies investigating sex steroids secretion in KS during infancy . UsuallySerum T concentrations tend to fall to the mid-low range in the young adult with KS , 76 in aOther reproductive hormones might be altered in KS. Serum estradiol might be almost normal or sometimes elevated, but the estradiol to testosterone ratio seems to be constantly higher than in normal men , 81 , comorbidities associated with KS such as diabetes, metabolic syndrome, osteoporosis and cardiovascular diseases usually appear during adulthood and increase with advancing age , 22, 89.When serum testosterone is below normal, obesity and gynoid fat distribution are common in men with KS , in addiTestosterone replacement therapy is effective in improving symptoms related to androgen deficiency, but not all other features related to the genetic abnormality Table . For thiSeveral other clinical features of KS have been related to hypogonadism, but with a variable degree of uncertainty. The finding of bone mineral density lower than normal is prevalent in patients with KS, but it seems not directly related to low serum testosterone . AccordiA case of a 51-year-old adult man affected by both KS and congenital adrenal hyperplasia (CAH) due to 21 hydroxylase deficiency, the first causing androgen deficiency, the latter leading to androgen excess was helpful in disclosing testosterone-dependent signs and symptoms in KS . Under-vPatients with a diagnosis of KS needs to be followed throughout life and have to be treated with testosterone in case of hypogonadism. Particular attention should be paid to adequate titration of testosterone dosage in these patients since they mostly have a mild testosterone deficiency, especially those with mild phenotype. All testosterone formulations are effective in patients with KS, the choice depending on the pretreatment levels of serum T, patients’ preferences and physicians’ attitude and experience with the formulations commercially available.Finally, the management of KS should also include the prevention or treatment of comorbidities.The main issue concerns how to improve a precocious diagnosis in order to reduce the number of patients who remains undiagnosed and to avoid the delay in the diagnosis. For these patients who are unaware of suffering of KS, it could be assumed that they are somewhat healthy and do not require medicalization and/or treatment. However, we do not know how many of them do not seek medical consultation for other reasons , but still complain of signs and symptoms of the disease. This gap in knowledge does not allow unraveling the entire spectrum of phenotypes of KS, a prerequisite useful to target and personalize the management of the disease, according with the real patient’s health status.In addition, some genetic aspects related to the phenotype remains undetermined. Despite the insights provided by numerous studies concerning the clinical consequences of KS performed so far, our knowledge of the molecular and cellular mechanism underlying the KS pathogenesis is still limited in part due to the lack of in-depth mechanistic studies. A part from the aneuploidy per se and the inter-individual genetic variation, several genetic mechanisms may play as other possible modulators of the variability of the phenotype observed in KS patients. It deals with the dosage effect and the expression/inactivation status of the X chromosome genes, the presence of mosaicism, the number and the derivation  of supernumerary X chromosome/s, the activity of the genes located in the pseudoautosomal regions (PAR) of the sex chromosomes.Furthermore, we do not know, at present, how to manage paucisymptomatic KS patients that remains undiagnosed since our knowledge on their real health conditions is very poor.Hence, the impact of the disease during fetal life and the early period after birth  remains an open issue. Furthermore, data on how patients with KS aging are scanty as well as information on how managing these patients in the elderly.Clinical and genetic phenotype of KS as well as their relationship are still not completely understood and need to be fully elucidated in order to improve also the clinical management of this disease.Accordingly, KS remains largely underdiagnosed (only 25 % of the expected number of patients are correctly diagnosed and only a minority before the puberty onset) and the majority of patients are often diagnosed during adulthood. Prompt educational and psychological supports might prevent any difficulties in their language, scholastic and neuropsychological difficulties; the start of the testosterone replacement therapy as soon as the patients need it allows them to avoid the long-term consequences of the hypogonadism; semen or testicular tissue cryopreservation could also be performed as soon as possible, before the testicular damage starts, probably at puberty. Indeed, a major effort should be done in order to increase our ability to perform early diagnosis of the KS and to provide advancement on the knowledge of both pathogenetic and clinical issues.


Vibrio vulnificus is a bacterium responsible for severe gastroenteritis, sepsis and wound infections. Gastroenteritis and sepsis are commonly associated with the consumption of raw oysters, whereas wound infection is often associated with the handling of contaminated fish. Although classical virulence factors of this emerging pathogen are well characterised, there remains a paucity of knowledge regarding the general biology of this species. To investigate the presence of previously unreported virulence factors, we applied whole genome sequencing to a panel of ten V. vulnificus strains with varying virulence potentials. This identified two novel type 6 secretion systems (T6SSs), systems that are known to have a role in bacterial virulence and population dynamics. By utilising a range of molecular techniques and assays we have demonstrated the functionality of one of these T6SSs. Furthermore, we have shown that this system is subject to thermoregulation and is negatively regulated by increasing salinity concentrations. This secretion system was also shown to be involved in the killing of V. vulnificus strains that did not possess this system and a model is proposed as to how this interaction may contribute to population dynamics within V. vulnificus strains. In addition to this intra-species killing, this system also contributes to the killing of inter bacterial species and may have a role in the general composition of Vibrio species in the environment. V. vulnificus is a Gram negative opportunistic pathogen typically isolated from marine and coastal environments ..V. fluviV. vulnificus is predominantly involved in anti-bacterial killing rather than anti-eukaryotic targeting, we investigated the role of T6SS1 in a Galleria melonella infection model. Groups of 10 larvae were injected with either, V. vulnificus 106-2A wild-type, SRC1 or SRC2 at 105 cells in the upper most pro-leg and monitored for signs of disease. The results however demonstrated that both SRC1 and SRC2 were as virulent compared to the wild-type strain, V. vulnificus for virulence in this infection model, further supporting our hypothesis that the T6SS1 of V. vulnificus is involved in modulating the composition of environmental bacterial populations.To further support our hypothesis that the T6SS1 of Vibrio species, this is the first report, to our knowledge, of a functional T6SS in V. vulnificus. Our study has shown a variation in the number of T6SSs present in ten different V. vulnificus isolates, with some having two T6SSs and others only one. This in contrast to V. cholerae, where numerous strains have been shown to possess just one conserved T6SS [V. vulnificus strains studied contained the T6SS, T6SS2 with a subset of strains containing an additional T6SS, T6SS1. Bioinformatic analysis of both T6SSs of V. vulnificus demonstrated that these systems contained the full repertoire of genes required for a functional secretion system [V. vulnificus T6SS2 contained accessory genes encoding for proteins such as a phosphatase and kinase, which have previously been shown to be required for T6SS regulation in P. aeruginosa [vasH. This gene encodes for an alternative sigma54 factor, which has previously been shown to be involved in initiation of transcription of the T6SS in V. cholerae [V. vulnificus.Although T6SSs have been characterised in many ved T6SS . All V. n system . V. vulnruginosa . Althougcholerae , 29 and V. vulnificus exhibits considerable overall synteny with the T6SS of V. cholerae [hcp and vgrG genes. For example, V. cholerae contains two hcp and three vgrG genes, in contrast to the apparent presence of only one copy of each in the V. vulnificus T6SSs. VgrGs play a structural role in the formation of the T6SS, however several vgrGs have also been shown to encode for effector functions, such as those identified in V. cholerae, and as such are denoted as evolved VgrGs [vgrGs are distinguishable from structural vgrGs based on the presence of a C-terminal extension. The vgrG genes in T6SS1 and T6SS2 of V. vulnificus appear not to be evolved vgrG genes due to the lack of a C-terminal extension.T6SS1 of cholerae . Howevered VgrGs , 24. EvoV. vulnificus is functional. Expression and secretion of Hcp-1 was shown to be affected by both temperature and salinity as has been observed previously in both V. cholerae and V. parahaemolyticus [V. vulnificus Hcp-1 was identical to those of Hcp-2 in V. parahaemolyticus [V. vulnificus Hcp-1 relative to V. parahaemolyticus Hcp-2 [Vibrio species is not uncommon, as V. cholerae V52 Hcp has been shown to be secreted at 37°C but Hcp of the O1 V. cholerae strain, A1552, is secreted at only 23°C [V. vulnificus T6SS1 is unlikely to be associated with an anti-eukaryotic phenotype. A hypothesis supported by our observation that SRC1 displays the same virulence in a G. mellonella model of infection to that of the wild-type.In this paper we show for the first time that T6SS1 of olyticus , 19. Ourolyticus . In contus Hcp-2 . Differenly 23°C . Our obsV. cholerae, [V. vulnificus T6SS1 may exhibit anti-bacterial properties. Furthermore, our observation that only some strains of V. vulnificus contain this additional T6SS1 led us to propose that this system may convey a competitive advantage. Through the use of co-culturing assays this hypothesis was supported by the observation that T6SS1 positive strains do have a competitive advantage over those that are T6SS1 negative. Furthermore, mutagenesis studies demonstrated that this phenotype was attributed to T6SS1 and not T6SS2.Based on T6SS1’s temperature regulation and similarity with the anti-bacterial T6SS of holerae,  we hypotV. vulnificus T6SS1, it is tempting to speculate on this system’s role in the natural environment, and potentially more widely in terms of clinical impact. Taken together, we hypothesise that the limited number of serious V. vulnificus human infections compared with the high abundance of this pathogen [V. vulnificus strains. This hypothesis is based on data in this study which has shown that a lesser virulent V. vulnificus strain can target a hyper virulent strain in a T6SS1 dependent manner, leading us to suggest that the same scenario may also occur naturally in the environment. This proposed scenario would potentially lead to a decrease in the number of hyper virulent strains in the environment, which may lessen the chances of humans being exposed to hyper virulent strains, and as such only a limited number of serious cases reported each year. This scenario is appealing as our study identified a predominance of T6SS1 among lesser virulent environmental strains [V. cholerae allowing for differing strains of V. cholerae to outcompete each other based on their effector repertoire [in silico investigations into the V. vulnificus T6SS1 and T6SS2 effectors has demonstrated that V. vulnificus contains mainly rhs effectors with DNase activity. However, to fully answer our proposed hypothesis the identification of V. vulnificus T6SS1 and T6SS2 effectors and immunity proteins needs to be elucidated, a study which our laboratory is currently undertaking.Given the observed antagonistic role of pathogen  may be a strains . In acco strains , 31. Howpertoire  this hypV. cholerae and S. marcescens utilise their T6SS for inter-species targeting [V. vulnificus T6SS1 could target other bacterial species. To test this hypothesis, co-culture assays were performed using the prey strain, V. fluvialis NCTC 11327. Both the anti-bacterial properties of T6SS1 and T6SS2 were investigated as it was hypothesised that T6SS1 may be specific to intra-species targeting and T6SS2 inter-species targeting. However, the results demonstrated that T6SS2 played no role in inter-species targeting. The finding that T6SS2 plays no role in anti-bacterial killing is not unusual as bacteria containing multiple T6SSs can often utilise these system for distinct roles. For example, although B. thailandensis contains a total of five T6SS, only T6SS-1 has been shown to be anti-bacterial, while T6SS-5 has been shown to possess an anti-eukaryotic phenotype [V. vulnificus T6SS2, it is likely that this system is tightly regulated. This hypothesis is based on the finding that T6SS2 contains the accessory proteins, phosphatase and kinases, two proteins which have been shown to be involved with tight regulation of the T6SS, HSI-I in P. aeruginosa [As well as intra-species targeting, bacterial species such as argeting , 32. Thehenotype . AlthougV. vulnificus T6SS1 mutant, SRC1 was unable to survive in the presence of V. fluvialis NCTC 11327 at 37°C. Suggesting that V. vulnificus requires T6SS1 for survival in mixed bacterial populations. Based on these findings, WGS and annotation was performed to determine whether V. fluvialis NCTC 11327 contained a T6SS which may account for the antagonistic manner of this strain. These investigations identified the presence of a T6SS locus in V. fluvialis NCTC 11327. Although the functionality of this system was not investigated, the T6SS locus contains all of the 13 genes required for a functional T6SS [V. cholerae V52 [V. fluvialis strain, 85003 also identified the presence of a T6SS cluster, suggesting that the T6SS is common among V. fluvialis strains [Vibrio family is not surprising as many Vibrio spp inhabit similar niches, therefore containing a mechanism such as the T6SS which can displace competing bacteria when resources such as space and nutrients are limited would be beneficial [During co-enumeration on TCBS agar, the nal T6SS  and is serae V52 . Similar strains . The widneficial .in vivo [V. vulnificus disease. For example, V. vulnificus and V. fluvialis share the oyster as a common niche and human infection route [V. fluvialis can target V. vulnificus. Furthermore, we have identified that the majority of V. vulnificus strains are T6SS1 negative. Therefore, we propose that upon human ingestion of an oyster containing V. fluvialis and T6SS1 negative V. vulnificus cells, V. fluvialis may target and remove V. vulnificus. In comparison to V. vulnificus which can exhibit varying degrees of pathogenicity [V. fluvilais is commonly associated with mild gastroenteritis [V. fluvialis could acts as a “probiotic”, by targeting and removing hyper virulent V. vulnificus cells, leaving only the lesser virulent V. fluvialis for the host’s immune system to eliminate. Based on this hypothesis, it is tempting to conclude that only a limited number of serious V. vulnificus infections would be reported each year. Further work is required to understand whether the removal of V. vulnificus by V. fluvialis is due to the V. fluvialis T6SS or whether there are potentially other diffusible anti-microbial molecules secreted by V. fluvilais. This is based on the finding that V. fluvilais was able to target V. vulnificus on enumeration plates where colonies were sufficiently diluted to not be in contact. It is highly likely that V. fluvialis may secrete diffusible anti-microbial molecules as other Vibrios have previously been shown to secrete anti-microbial molecules other than those associated with the T6SS [In addition to environmental attacks, a recent publication demonstrated the antagonistic behavior of the T6SS in vivo . The autin vivo . Similaron route , 23 and genicity , V. fluvthe T6SS .V. vulnificus. Furthermore, we have identified the conditions under which T6SS1 is active, and demonstrated that T6SS1 contains anti-bacterial properties. These findings have been used to hypothesise how V. vulnificus T6SS1 may be used to shape Vibrio microbial populations and how inter-bacterial interactions may explain the limited number of serious human infections attributed to V. vulnificus given the natural prevalence and virulence potential of this bacterium.In conclusion, this study has identified for the first time two T6SSs in V. vulnificus strains and V. fluvialis NCTC 11327, were routinely cultured at 30°C and V. choerlae V52 at 37°C in Luria-Bertani (LB) broth (Oxoid) unless otherwise stated. Where appropriate, antibiotics (Sigma) were added at the following concentrations; ampicillin (Amp) 100 μg/ml, Kanamycin (Km) 50 μg/ml, chloramphenicol (Cm) 35 μg/ml for Escherichia coli and 10 μg/ml for Vibrio vulnificus, Streptomycin 100 μg/ml and Trimethoprim (Tp) 100 μg/ml. Strains, plasmids and primers used in this study are listed in Tables V. vulnificus isolates was carried out by the Exeter Sequencing Service, University of Exeter, Exeter, UK. De-novo assembly of raw reads was performed using the A5-piepeline [Vibrio vulnificus S2-22 (LKUU00000000); Vibrio vulnificus 106-2A (LMTD00000000); Vibrio vulnificus ORL-1506 (LMXV00000000); Vibrio vulnificus NSV-5830 (LMXW00000000); Vibrio vulnificus 99–796 (LMXX00000000); Vibrio vulnificus 99–743 (LMXY00000000); Vibrio vulnificus DAL-79087 (LMXZ00000000); Vibrio vulnificus DAL-79040 (LMYA00000000); Vibrio vulnificus ATL-9824 (LMYB00000000); Vibrio vulnificus S3-16 (LMTC00000000); Vibrio fluvialis NCTC11327 (LMTE00000000).WGS of iepeline  followediepeline . MappingV. vulnificus 106-2A were produced using pDM4 [E.coli DH5α and chromosomally introduced to V. vulnificus 106-2A using the conjugal donor pRK2013 and plated onto Cholera Medium TCBS (Oxoid) supplemented with choloramphenicol at 10 μg/ml. Meridiploid strains were confirmed using LFF or RFR flanking primers with cmpDM4_F or cmpDM4_R primers. Chromosomally confirmed first cross-over integrants were then plated onto LB supplemented with 10% sucrose (Sigma) incubated at 24°C to exercise the plasmid in a second cross-over event replacing the wildtype gene with the mutated gene. Mutants were confirmed using MutScr primers detailed in SRC1 and SRC2 mutants of ing pDM4 . FlankinAntibiotic resistant strains were generated using tri-parental conjugal mating using the helper strain pRK2013 and plated onto TCBS agar containing appropriate antibiotic.V. vulnificus cultures were back-diluted to an OD590 0.03 in 25 ml volumes and grown to mid-log OD590 1.5. Cellular fractions were normalised to OD590 1 in 1ml LB, centrifuged at 2000 rpm for 3 min, supernatant removed and pellets re-suspended in 50 μl bug buster, 25 μl PBS and 25 μl 4x loading buffer and heated at 100°C for 10min. Supernatants were filter sterilised using a 0.2 μm filter and proteins precipitated with 100% TCA on ice for 45mins and centrifuged at 20,000 g for 5mins at 4°C. Pellets were washed with acetone, heat dried at 95°C and re-suspended in sterile PBS. Precipitated proteins and cell pellets were quantified using the BCA assay (Thermo Fisher) and equal protein loadings were subjected to SDS-PAGE and Western blotting using primary Hcp-1 antibody [Overnight antibody  (1:500) Vibrio strains were adjusted to OD590 0.03 and grown to OD590 ~1.00, cultures were then re-adjusted to OD590 0.8. Attacker and prey strains were then mixed at a ratio of 3 mls: 1 mls respectively. 200 ml of these mixed cultures were then incubated at 30°C for 5 hours on LB plates to allow contact dependent-killing in triplicate. Following incubation, co-cultures were harvested from the plates and re-suspended in 1 ml of sterile PBS for enumeration. 10-fold serial dilutions of these suspensions were prepared and 10 μl of each dilution was spotted onto TCBS agar plates in triplicate. These enumeration plates were then incubated at 37°C overnight. The average cfu/ml was recorded for each triplicate dilution.Overnight cultures of V. fluvialis NCTC 11327 as prey were incubated on LB plates at 30°C for five hours and co-cultures using V. vulnificus MO6-24/O as prey were incubated on LB plates at 30°C for two hours. Following incubation to determine T6SS dependent killing, co-cultures were harvested from the plates and re-suspended in 1 ml of sterile PBS for enumeration. 10-fold serial dilutions of these suspensions were prepared and 10 μl of each dilution was spotted in triplicate onto LB agar plates that contained trimethoprim and onto another set of LB plates containing chloramphenicol. These enumeration plates were then incubated at 37°C overnight. The average cfu/ml was recorded for each triplicate dilution.Assays were performed as described above with the following modifications: prey and attacker strains were grown overnight in LB containing chloramphenicol (10mg/ml) and trimethoprim (100 mg/ml) respectively. Prior to mixing of the prey and attacker strains, overnight cultures were centrifuged and re-suspended in fresh LB media containing no antibiotic to prevent killing due to the antibiotics. Co-cultures using G. mellonella weighing between 2.0 and 3.0 g were injected in the upper most right proleg with V. vulnificus cells that had been grown overnight at 37°C in LB using a 25 μl Hamilton Microlitre™ syringe 800 series with removable needle (Sigma). Overnight bacterial cultures were adjusted to ~ 1x107 cfu/ ml (equating to ~ 1x105 cfu/10 μl). Enuermation of the infective dose was also performed to ensure the correct bacterial input. Infected larvae were then incubated at 37°C and monitored for signs of infection, death was recorded when larvae failed to respond to touch.S1 FigBoxed maps are those of previously sequenced reference strains.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


This perspective describes the construction of 0D–3D organic and polymeric architectures using “robust” supramolecular scaffolds. To assemble functional molecular units into a desired structure while controlling positional and orientational order is a key technology for the development of high-performance organic materials that exhibit electronic, optoelectronic, biological and even dynamic functions. For this purpose, we cannot rely simply on the inherent self-assembly properties of the target functional molecular units, since it is difficult to predict, based solely on the molecular structure, what structure will be achieved upon assembly. To address this issue, it would be useful to employ molecular building blocks with self-assembly structures that can be clearly predicted and defined, to make target molecular units assemble into a desired structure. To date, various motifs of molecular assemblies, polymers, discrete and/or three-dimensional metal–organic complexes, nanoparticles and metal/metal oxide substrates have been developed to create materials with particular structures and dimensionalities. In this perspective, we define such assembly motifs as “supramolecular scaffolds”. The structure of supramolecular scaffolds can be classified in terms of dimensionality, and they range in size from nano- to macroscopic scales. Functional molecular units, when attached to supramolecular scaffolds either covalently or non-covalently, can be assembled into specific structures, thus enabling the exploration of new properties, which cannot be achieved with the target molecular units alone. Through the classification and overview of reported examples, we shed new light on supramolecular scaffolds for the rational design of organic and polymeric materials. Molecular self-assembly that proceeds under thermodynamic control is a useful approach to creating organic materials with high positional and/or orientational order of the constituent molecules.Nature uses superb scaffolds and achieves multi-component assembly into dedicated structures for a particular function, as represented by the deoxyribose–phosphodiester backbones of DNA and peptide backbones. Light-harvesting systems such as LH1 also involve a highly sophisticated scaffold.via non-covalent interactions can also be regarded as supramolecular scaffolds.60 units. By attaching to an appropriate supramolecular scaffold, C60 units can be assembled into zero-,To the best of our knowledge, the term “supramolecular scaffold” was first introduced by Hunter and coworkers in 1996 to describe a macrocyclic host molecule that can bind to both electron-donor and -acceptor molecules simultaneously.2.Zero-dimensional (0D) assemblies of functional molecular units have been designed to provide, for instance, efficient light-harvesting, multivalent molecular recognition, catalytic systems, and templates for the synthesis of discrete inorganic materials.16e.g., 1,3-bipyridylbenzene (1–5) and 1,3-bis(4-ethynylpyridyl)benzene derivatives (6–16)], when mixed with a Pd2+ source such as Pd(NO3)2, self-assemble quantitatively into hollow spherical cuboctahedral nano-objects consisting of 24 ligands and 12 Pd2+ ions ,2),3),4)5)1–5 can accurately assemble into an M12L24 structure, where the corresponding functional molecular units are located on the outer surface of the scaffold  with functional molecular units gives rise to inner space-functionalized M12L24 complexes (6),7),8),9),10),11),12),13),14),N-oxyl (TEMPO) radical (15)16)13) can provide a template for the synthesis of TiO2 nanoparticles.14, 15 and 16) show excellent molecular recognition and catalytic properties arising from the densely arranged functional units in the cavity of the M12L24 complexes.Meanwhile, pre-functionalization at the 2-position of the central benzene ring of 1,3-bis(4-ethynylpyridyl)benzene ligands  and four perylene bisimide-based bidentate ligands as a supramolecular scaffold for constructing electroactive and light-harvesting systems.Würthner and coworkers utilized square-shaped metal complexes consisting of four metal ions (Pt17) having imino groups at its branched positions 20),21)22)Au nanoparticle (AuNP) with a narrow size distribution is also useful as a 0D scaffold for thiol-containing molecules .16 A var3.Fibrous and tubular assemblies as well as linear polymers can provide one-dimensional 1D) supramolecular scaffolds .1–9,45–4D supramo23),24),25),26),27)60 (28)23, which serves as a component of a liquid crystal,26 undergoes a reversible sol–gel transition triggered by chemical redox reactions, sonication and a temperature change.50Naturally occurring cholesterol derivatives are well known to show particular self-assembling properties.trans-1,2-cyclohexanebisamide (29) assembles to form a fibrous assembly via intermolecular hydrogen-bonding between amide groups (30),31),32),33),34),35)36),30 provides a template for the formation of a TiO2 nanofiber in the hydrolysis-condensation of Ti(OiPr)4 in the organogel of 30 in EtOH/NH3 aq.2 nanofiber can be further converted into a hollow nanotube by removing the 1D scaffold through calcination. Similarly, Shinkai and coworkers successfully obtained a helical silica nanofiber by the condensation of tetraethoxysilane in an organogel of a mixture of 29 and ammonium-appended 31 in ethanol/water followed by calcination.32–34 on the 1D scaffold show enhanced photovoltaic response, carrier mobility and emission behaviour compared to their non-assembled states.35 shows high charge-transporting capability, with a charge-diffusion coefficient of 3.3 × 10–7 cm2 s–1 in acetonitrile.36 for the fabrication of organogel with a photochromic fluorescent-switching behavior.60Hanabusa and coworkers reported that enantiopure e groups .53 With 37 and optically active 38.39 and 40 (39 : 40 = 97.5 : 2.5) with an achiral phosphine unit and a chiral inducer, respectively.41),i) ions in the presence of a helical 1D scaffold composed of BTA derivatives with terpyridyl groups (42) and chiral acid moieties (43) resulted in the formation of a helical array of AuNPs, which showed significantly enhanced optical activities compared to the scaffold itself.44–48 can be used in intracellular gene delivery.3+ complex-appended BTA (49) and a peptide-appended BTA (50)Benzene-1,3,5-tricarboxamide (BTA)51–54, 51  and biotin (54) hybrids with an extended BTA scaffold have been used for bacteria-detection and protein-assembly, respectively.64The attachment of 2,2′-bipyridyl groups to BTA results in an extended version of the building blocks for a 1D scaffold 51–54, .61,64,711–54, 51  and TTF-i.e., its interior and outer surfaces, for the controlled assembly of functional molecular units.75A 1D tubular scaffold can provide two distinct sites, peri-hexabenzocoronene (HBC) derivatives 56)60-functionalized nanotube, by efficient electron transfer between HBC and the acceptors upon photoirradiation. A nanotubular assembly capable of switching photoconductivity has been realized by the self-assembly of HBC 57 with a pre-functionalized diarylethene unit.2+ complex, the organic radical-appended HBC (58) undergoes controlled assembly to form a nanotube having N,N-Co2+ coordinated copolymer chains on its inner and outer surfaces. Such coordinated copolymer chains can also be formed by post-functionalization of the self-assembled nanotube of 58. The former nanotube exhibited significantly higher magnetic susceptibility than the latter. HBC 59 self-assembles to form a nanotube whose inner and outer surfaces are densely covered by azide groups.60) allows site-selective post-functionalization, resulting in a nanotube with different functional units on its inner and outer surfaces.61) are completely dispersed in water, not as a bundle but as individual nanotubes.62), after pre-functionalization with a chiral Pt2+ complex, self-assembles to form enantio-enriched nanotubes in terms of the helical chirality of π-stacked HBC arrays.2+ complex. A nanotube formed from bipyridyl-appended HBCs (63) undergoes post-functionalization with a Cu2+ complex.84The use of ionic molecular units gives rise to a water-dispersible 1D tubular scaffold. For instance, nanotubes formed from isothiouronium ion-appended HBC  derivatives with a hydroxyl group at the 364–69 self-assemble into 1D fibres, which have many biological applications.13–C16) at the N-terminus of the peptide, a β-sheet-forming part, a charge-containing part, and a functional unit domain at the C-terminus (64) hybridized with a PA-based 1D fibril scaffold exhibits enhanced bioactivity compared with that in the non-assembled state.65 with a heparin-binding Leu-Arg-Lys-Lys-Leu-Gly-Ala (LRKKLGKA) unit, which serves as a charge-containing and functional domain  building blocks such as terminus .87,88 A 70),71),72),73),74, 75),76)77 and 78).80 has been achieved by the post-functionalization of polyacetylene 79.71),73 and 74)76).106Helical polymers are also extensively used as a scaffold for the construction of helical 1D assemblies of functional molecular units.81),82),83),84),85)86)82 with an n-type semiconducting unit provides a component for a thin-film transistor device. Strikingly, it has been demonstrated that a photovoltaic cell fabricated with 82 as an n-type active layer exhibits a 20-fold higher overall conversion efficiency (0.2%) than that fabricated with the corresponding perylenediimide monomer.87 and 88) or catalytically active groups (89 and 90) without impairing the degree of its one-handedness,Polyisocyanides are an interesting class of helical polymers that allow the very high-density assembly of functional molecular units upon covalent pre-functionalization.Naturally occurring polymeric materials such as duplex DNA have also been used as a helical 1D scaffold.4.Supramolecular scaffolds that enable controlled two-dimensional (2D) assembly  are rela12491–94,125Recently, we demonstrated the rational synthesis of organic thin films with exceptionally long-range structural integrity using tripodal paraffinic triptycene building blocks 91–94,.22,125–195–100) have also been developed.97 and 99 possess a large dipole moment along the C3 axis of triptycene due to the presence of an electron-withdrawing cyano or dicyanovinyl group on the opposite side of an electron-donating hydroxyl or alkoxy group. Interestingly, these dipolar tripodal triptycenes change the insulator-to-metal transition temperature of VO2 thin films upon surface adsorption.101–106 have been designed, which offer sufficient space to accommodate functional molecular units with a size comparable to the diameter of the triptycene framework . Notably, when a functional molecular unit is sandwiched between the cationic and anionic peptoids, it periodically aligns in the resulting 2D sheet  with an alternating sequence of rigid π-conjugated parts and hydrophilic ethylene glycol parts  became fluorescent.113) has been reported to exhibit similar folding-assembling behaviour in solution to form a 2D sheet.134Ikeda and coworkers developed a polymer  or porous coordination polymer (PCP), can be regarded as a typical three-dimensional (3D) scaffolds. So far, a huge number of porous crystals have been developed using directional coordination bonding between metal ions and organic ligands .140–143 2+ and Zn2+) can incorporate and precisely arrange guest molecules such as TTF and C60 in the pore upon immersion of the crystals in a solution of the guest molecule (Recently, an interesting application of porous crystals has been reported by Fujita and coworkers. Porous crystals consisting of tripyridyltriazine ligands and metal ions (Comolecule .25 With The realization of highly oriented large-area 3D assembly is one of the key issues in the development of high-performance materials and devices. Fukushima, Aida and coworkers found that bottle-brush polymers, which carry side chains with three mesogenic cores, can form a completely oriented, large-area 3D ordered structure by a simple one-step hot-press treatment between Teflon sheets .151,152 6.The concept of a supramolecular scaffold, which originally represented a molecular motif that directed the formation of a discrete supramolecular complex in solution, has been extended to self-assembling objects with well-defined dimensionality at various length scales. Target functional molecular units, upon pre-functionalization to the building block of a supramolecular scaffold or post-functionalization to a supramolecular scaffold, undergo controlled assembly into a structure that reflects the morphology and size regime of the supramolecular scaffold used. In principle, through the use of a supramolecular scaffold, any molecular unit, regardless of its inherent self-assembly properties, may assemble into a purposely designed structure. From the perspective of the transcription of structural information, the concept of a supramolecular scaffold is analogous to that of a conventional “template”, while the former more strongly aims to create new functionalities through, for example, interactions between the scaffold and incorporated molecular units or interactions among molecular units assembled on/in the scaffold. As exemplified by chiral amplification effects, a high-density assembly of functional molecular units might lead to a non-linear responsive behaviour. The use of supramolecular scaffolds might bring about a metastable assembly state of a target molecular unit. Unlike a thermodynamically stable state, such a metastable assembly might be susceptible to external stimuli, and thereby could exhibit unusual responsive behaviours. Through the elaborate design of supramolecular scaffolds, it may be possible to assemble multiple components precisely into a structure with a periodic order. Self-assembly motifs that are suitable for application as a supramolecular scaffold are being developed. Therefore, we expect that the concept and range of application of supramolecular scaffolds may further expand to more diverse molecular systems to promote the rational design of organic and polymeric functional materials as well as the discovery of unknown molecular behaviours.There are no conflicts to declare.


The VRC nanofibers were characterized by infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The proton nuclear magnetic resonance (1H-NMR) was used to analyze the molar ratio of HPβCD/VRC in the nanofibers. Compared with a VRC solution, the nanofibers significantly prolonged the half life, and increased the bioavailability of VRC in rabbit tears. No obvious signs of irritation were observed after application in the conjunctival sac. VRC nanofibers are promising for ophthalmic drug delivery and further pharmacodynamics studies are needed.Voriconazole (VRC) incorporated in composited polyvinyl alcohol (PVA)/hydroxypropyl-β-cyclodextrin (HPβCD) blended nanofibers were produced via electrospinning for efficient ophthalmic delivery. The VRC loading capacity increased with increasing HPβCD content. The optimal solution for electrospinning consisted of 8% (w/v) PVA, 4% (w/v) HPβCD and 0.5% (w/v) VRC. The nanofibers exhibited bead-free average fiber diameters of 307±31 nm and VRC was released  Fungal keratitis, a fungal infection of the cornea, is one of the most serious ocular fungal infections. With the extensive application of broad-spectrum antibiotics and corticosteroids, the incidence of fungal keratitis has increased recently. It has become the major cause of blindness caused by ocular fungal infections , 2. The Voriconazole (VRC), a triazole antifungal agent, is a lipophilic drug LogP 1.65) with low aqueous solubility (0.5 mg/ml in distilled water). It possesses promising characteristics such as broad-spectrum activity, activity against resistant fungal species, good oral bioavailability, and acceptable tolerability. It is highly effective against various fungal isolates associated with keratitis , 5. It i with lowin vitro drug release, biocompatibility and pharmacokinetics in rabbit tears were investigated. The VRC nanofibers are safe for ocular use and they can significantly increase the half life and bioavailability of VRC in tears.Electrospinning is a versatile and cost-effective method for producing nanoscale fibers from polymer solution or polymer melts with the help of a very strong electric field. Nanofibers have received great attention in tissue engineering, enzyme immobilization and drug delivery systems in recent years , 16. Thecetonitrile  of HPLC grade were used. Male New Zealand white rabbits weighting 2.5–3.0 kg were purchased from Zhejiang Academy of Medical Science . The rabbits were bred in normal way and all the animal experiments were performed in full compliance with guidelines approved by the Animal Care Committee at Zhejiang University City College. All efforts were made to minimize suffering.Polyvinyl alcohol (PVA AH-26) was supplied by Zhejiang Changqing Chemical Co., Ltd. VRC was purchased from Huahai Pharmaceutical Co, Ltd. . HPβCD from Fengyuan Biotechnology Co., Ltd  and The solubility study was performed according to the method reported by Higuchi and Connors . Excess morphology were determined. Finally, drug loaded spinning solutions were prepared by mixing PVA solutions with VRC dissolved in a HPβCD solution at 50°C. The finally solutions consisted of 8% (w/v) PVA, 4% (w/v) HPβCD and 0.5% (w/v) VRC (F1), 8% (w/v) PVA, 6% (w/v) HPβCD and 0.7% (w/v) VRC (F2), or 8% (w/v) PVA, 8% (w/v) HPβCD and 0.8% (w/v) VRC (F3).To determine the optimal concentration of PVA for the electrospinning process, 6%, 8% and 10% (w/v) PVA were used to prepare blank nanofibers. Subsequently, varied amounts of HPβCD were added to the polymer solution to achieve the final concentration of 4%, 6% and 8% (w/v). The effects of cyclodextrins on nanofibers The mixed solutions were then placed into a 20 mL plastic syringe equipped with a flat-tipped stainless steel needle (d = 1.2 mm). An emitting electrode of positive polarity from a high-voltage DC power supply  was connected to the spinning solution. A grounded copper plate was used as a collector. The electrostatic field strength was fixed at 18 kV/20 cm. The feed rate of the solution was controlled to 0.5mL/h using a syringe pump . For the morphological study, the collection time was approximately 5 min, while, for the rest of the experiments, the collection time was 6 h. The collected nanofibers were placed in a vacuum oven overnight at 40°C to eliminate residual solvent.The thickness was measured with a hand-held micrometer . The values were determined at five different regions of the film.The morphological appearance of the nanofibers was assessed using a scanning electron microscope . The average diameter of the fibers was evaluated using Image J software by measuring 100 fibers from the SEM images.1H-NMR) spectra were recorded at 400 MHz . 5 mg VRC, 10 mg PVA/HPβCD nanofibers or 10 mg VRC nanofibers were dissolved in 0.5 mL d6-DMSO.The proton nuclear magnetic resonance  analyses were performed for VRC, HPβCD, PVA, PVA/HPβCD nanofibers and VRC nanofibers. TGA was conducted under nitrogen atmosphere by heating the samples from 50 to 600°C at the heating rate of 20°C/min.A differential scanning calorimeter  was used to investigate the thermal behavior of the VRC nanofibers. The DSC thermograms  were obtained during heating from 35 to 260°C at a rate of 10°C/min.-1.The chemical structures of VRC nanofibers and PVA/HPβCD nanofibers were characterized using a Fourier transform infrared spectrophotometry  with the accessories of attenuated total reflectance (ATR). VRC were ground and pressed into KBr dishes pellets before analysis from 400 to 4,000 cmacetonitrile. A VRC solution was used as a control.Nanofiber films containing 3 mg of VRC were immersed in 100 mL of 0.9% NaCl at 32°C with shaking at 50 rpm. The release medium was sampled (1 mL) for the drug assay and an equal amount of fresh medium was added at the times indicated. Before the HPLC assay, the samples were diluted with Eight rabbits were separated into a nanofiber-treated group and a VRC solution-treated group. VRC solution was prepared by dissolving VRC in a 0.9% (w/v) HPβCD solution to achieve a final 0.15% (w/v) drug concentration.Rabbits were treated with a single administration of 40 μL of VRC solution or 1.5 mg of nanofibers, in which the drug dose was equivalent to that of the VRC solution. Tear fluid samples were collected at 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, and 24.0 h after administration in the nanofiber group and at 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0, and 8.0 h after administration in the VRC solution group, using filter papers, which were weighed before use and gently inserted into the inferolateral cul-de-sac of the eye, close to the middle of the eye, for 30 seconds. After collection, papers were weighed again. Then, the papers were dried under stream of nitrogen gas and vortexed with 500 μL of methanol for 5 min. After centrifugation  for 10 min, the organic phase was transferred to another tube and evaporated to dryness. The residue was reconstituted with 50 μL methanol. The drug concentration in each sample was determined using HPLC.An ocular irritation test (Draize eye test) was used to evaluate the ocular tolerance of the nanofibers.Eight rabbits were randomly divided into two groups. One eye of the rabbit was treated with the VRC solution or VRC nanofibers, while the untreated eye functioned as a control. A successive administration once a day for 7 days was performed at the same dosages as those used in the pharmacokinetic study. The ocular irritation was observed at 1, 8 and 24 h after administration. The animals’ discomfort and symptoms in the conjunctiva, cornea, and lids were macroscopically examined using the Acute Eye Irritation/Corrosion scoring system established by the 2012 Organization for Economic Cooperation and Development for ocular irritation testing  26]..26].At the end of the test, the rabbits were euthanized by injection of a lethal dose of pentobarbital into the marginal ear vein. The cornea tissues were collected and fixed by immersion in buffered formalin (pH 7.4), dehydrated and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) to evaluate potential injury to the cornea.MVRC is the actual VRC content in the weighed quantity of nanofibers, and MNF is the weighed quantity of nanofibers.The drug content in the VRC nanofibers was quantified by dissolving them in 30% (v/v) ethanol. The solutions were analyzed using HPLC. Drug loading rate (DL) was calculated:18 column; flow rate: 1.0 mL/min; the UV detector wavelength: 256 nm. The mobile phase was acetonitrile: water (46:54).The HPLC system used was of the Agilent 1260 series  and detection conditions were as follows: Agilent ZORBAX SB-CP-value of 0.05 was considered to be significant.Kinetica™ V.4.4 with a non-compartment model was utilized to calculate the pharmacokinetics parameters in rabbit tears. Statistical data were analyzed using a Student’s t-test and a L type, according to Higuchi and Connors, indicating the formation of soluble complexes of a 1:1 stoichiometry [Kc) value was estimated to be 320 L/mol, according to the following equation [linear regression of the profile and C0 was the solubility of VRC in H2O. Compared with sulfobutylether-β-cyclodextrin (SBE-β-CD) used in VRC injections [HPβCD, a cyclic oligosaccharide with an outer hydrophilic surface and a lipophilic cavity, is the most thoroughly studied cyclodextrin derivative. Numerous toxicological studies, pharmaceutical technological experiments and human clinical trials have been performed with HPβCD . It is chiometry . The appequation :Kc=slopjections , HPβCD bmorphology , few tolerance problems should be expected after application in the conjunctival sac [Cylindrically shaped continuous nanofibers without non-connected ‘tailed’ beads were formed C1. The aival sac .1H-NMR) was employed to further explore the molar ratio of HPβCD/VRC in nanofibers (1H-NMR (d6-DMSO), δ (ppm): 9.05 , 8.24 , 8.08 , 7.63 , 6.95–6.91 , 2.51–2.5 , 1.11  and HPβCD: 1.03  were showed in the spectra of VRC nanofibers. The results indicated that VRC as well as HPβCD were electrospun in the nanofibers. In order to make the molar ratio calculations, the integration of HPβCD and VRC peaks were used. The molar ratio of HPβCD: VRC in nanofibers was calculated as 1:0.54 by taking the integration of the protons of HPβCD at 1.03 ppm and VRC at 1.11 ppm. According to the actual drug loading rate determined by HPLC, the molar ratio was calculated as 1:0.5. 1H-NMR results was in good consistence with HPLC results. Substantial amount of VRC was incorporated in nanofibers during the preparation and electrospinning.Proton nuclear magnetic resonance  for PVA was 218.42°C obtained from the DSC scan of PVA powders. Two endothermic peaks at around 120 and 215°C corresponding to the characteristic peaks of HPβCD and PVA were observed in PVA/HPβCD nanofibers. The thermogram of the VRC powder exhibited a melting point at 132.44°C whereas no melting point was observed for the VRC nanofibers, suggesting that VRC was incorporated into nanofibers in an amorphous state.DSC studies were undertaken to evaluate the physical state of VRC in the electrospun nanofibers and the thermograms are shown in -1 that are attributed to the stretching vibrations of OH, CH and CO, respectively [-1 corresponding to the stretching vibrations of OH. The bands at 3000–2850, 1600–1400, and 1360–1250 cm-1 were assigned to the alkane CH, C = C aromatic and aryl C-N stretches [The FT-IR spectra of the VRC powder, PVA powder, HPβCD powder, PVA/HPβCD nanofiber and VRC nanofiber are displayed in ectively . The VRCtretches . The domtretches . There wtretches . During tretches . Howeverin vivo analytical requirements. The low limit of quantification and low limit of detection of the method were 0.1 μg/mL and 0.03 μg/mL, respectively. The extraction recoveries at low, medium and high VRC levels were all greater than 90%. The standard curve for the drug assay in rabbit tears was Y = 418921X-21369 within 0.1–10 μg/mL VRC. The standard curve showed good linearity and the R2 was 0.9991.The majority of ophthalmic treatments are delivered as highly concentrated eye drops. They suffer from short precorneal residence time which is associated with low corneal drug absorption. Nasolachrymal drainage, lacrimal replacement and the relative impermeability of the corneal epithelial membrane lead to rapid and extensive loss of drugs from the precorneal area . Therefoin vitro. The calculated pharmacokinetic parameters are listed in t1/2 and AUC0-t. The nanofibers significantly prolonged the VRC half-life in tears, and increased the VRC bioavailability. Compared with the VRC solution, the relative bioavailability was 245%. The interaction of HPβCD with biological membranes has been reported to facilitate drug absorption in the tear film [The drug tear concentration-time profiles of the two formulations are shown in ear film . In addiPVA, which formed the nanofiber matrix, is a well-tolerated polymer type for ophthalmic drug delivery systems , 44. HPβThe Draize eye test results show that there was no ocular discomfort after the administration of the VRC nanofibers and the VRC solution . AccordiAUCt0→, and bioavailability of VRC were all significantly increased in rabbit tears. Furthermore, few tolerance problems were observed after consecutive application for 7 days. It can be concluded that these nanofibers represent a promising delivery system to prolong the ocular delivery of VRC.In this research work, we successfully developed VRC incorporated PVA/HPβCD nanofibers for the treatment of fungal keratitis. The physicochemical characteristics, pharmacokinetics and ocular irritation of the optimized fibers were evaluated. SEM, TGA, DSC and FT-IR analyses suggest that VRC is present in an amorphous state in the nanofibers and there are interactions between VRC and HPβCD. Drug loading was increased because of the solubilization effect of HPβCD. A sustained release of VRC for 24 h was achieved. The half-life,


Vibrio vulnificus can cause severe disease in humans who consume undercooked, contaminated seafood. To study food-borne V. vulnificus disease in the laboratory, mouse virulence studies predominantly use death as the primary experimental endpoint because behaviorally based moribund status does not consistently predict lethality. This study assessed ventral surface temperature (VST) and its association with mouse survival during V. vulnificus virulence studies as an efficacious, humane alternative.The Gram-negative bacterium V. vulnificus was measured every 2-h for 24 h and data for minimal VST analyzed for prediction of lethal outcome.VST of mice intragastrically inoculated with V. vulnificus exhibited hypothermia with minima occurring 8 to 12 h post-inoculation. The minimum VST of mice that proceeded to death was significantly lower than that of surviving mice. VST ≤ 23.5 °C was predictive of subsequent death with a sensitivity of 68% and specificity of 95%.In contrast to the relatively stable VST of mock-infected control animals, mice infected with V. vulnificus infection has potential to reduce suffering of nearly 70% of mice for a mean of 10 h per mouse, without compromising experimental efficacy. Temperature cutoff of 23.5 °C exhibited 93% positive and 77% negative predictive value. For future V. vulnificus virulence studies requiring only binary comparison , we find that VST can be applied as a humane endpoint. However, use of VST is not recommended when detailed survival kinetics are desired.Use of VST ≤ 23.5 °C as an experimental endpoint during  Vibrio vulnificus is a Gram-negative bacterium found in aquatic environments. It has the potential to cause disease in humans who encounter the bacterium [V. vulnificus infections with mortality rates exceeding 50% of infected individuals [V. vulnificus pathogenesis is increasingly critical.acterium , 2. Wounacterium . Gastroiacterium . In seveacterium . Survivaividuals . In lighV. vulnificus treatment efficacy, and retrospective clinical analyses lack much of the detailed information necessary to obtain full comprehension of disease course [V. vulnificus foodborne infection, a mouse model is routinely employed [Controlled human case studies cannot be conducted due to low e course –11. Theremployed , 13. Folemployed , 14, 15.V. vulnificus mortality after oro-gastric inoculation, and to define the specific temperature parameters for implementation of hypothermia as a humane endpoint in V. vulnificus intragastric virulence studies.While laboratory mouse experiments provide invaluable data on infectious diseases, animal distress is, in many cases, an inherent component of animal experimentation. For this reason, scientists as well as regulatory bodies have sought methods to limit suffering of murine research subjects . CurrentV. vulnificus that differ in the gene rtxA1, which encodes the primary virulence toxin, the Multifunctional Autoprocessing Repeats-in-Toxins (MARTX) toxin. All strains are detailed in Table These experiments were conducted using four strains of V. vulnificus strain HG0902 (CMCP6rif ΩvvhA::bla) was generated from the Korean clinical isolate V. vulnificus CMCP6rif. Although originally described as a genetic deletion of vvhBA [vvhA by the stable integration of a portion of ampicillin-resistant plasmid pHGJ4. This strain was then further modified. Plasmids for alteration of rtxA1 to rtxA1::bla, mcf::bla, or Δmcf have been described previously [Escherichia coli SM10λpir or S17λpir to V. vulnificus HG0902 by conjugation. Selection for double homologous recombination was conducted using sucrose counterselection to isolate recombinants as previously described [of vvhBA , subsequeviously , 22, 23.escribed . GeneticV. vulnificus was streaked from frozen glycerol stocks onto LBrif plates 2 days prior to experimentation. The following day, single colonies were picked and grown in 2 mL LB broth with selective antibiotic overnight at 30 °C with shaking at 225 rpm. The following morning, strains were subcultured 1:100 into LB without antibiotic and grown to mid-log phase at 30 °C with shaking at 225 rpm. Cultures were pelleted and resuspended in sterile phosphate buffered saline  to indicated concentrations based on optical density (A600). Bacterial inoculum concentration was confirmed by dilution plating.Bacteria were routinely grown in Luria-Burtani (LB) broth  containing 50 μg/mL rifampin as needed. For all experiments, ad libitum. Experiments were conducted when mice were 5–6 weeks of age. For each of the two independent experiments, 25 mice were divided into 5 groups of 5 mice each to be either mock-inoculated with PBS, or inoculated with one of the four V. vulnificus strains.Fifty female C57BL/6 mice were obtained from Jackson Laboratories . Animals were housed 5 per cage in wood shavings with disposable paper huts and cotton bedding squares as nesting material. Water and food were provided 8 colony-forming units (CFU) V. vulnificus suspended in PBS using a 1-cm animal feeding needle attached to a 1-mL syringe. Mice were then injected intraperitoneally with 100 μL of a cocktail containing 10 μg/ml ketamine and 2 μg/ml xylazine in PBS to facilitate bacterial infection [Each mouse was transiently anaesthetized using isoflurane and then inoculated intragastrically (i.g.) with 50 μL of PBS or 1 × 10nfection .Mice were monitored hourly for the first 24 h (h) and subsequently every 4–8 h until 48 h post-infection (hpi). Ventral surface temperature (VST) was measured every 2 h during the first 24 h then once at 40–42 h and once at 47–48 h. Temperature was measured using the non-contact infrared TW2 thermometer  as recommended by the manufacturer. Thermometer emissivity maintained at the manufacturer’s default setting of 0.95. The TW2 thermometer is accurate +/− 1.0 °C when the object of interest is between 15 and 35 °C.To obtain temperature measurements, mice were restrained by scruffing. The thermometer was held approximately 6 cm from the mouse ventral side and the beam aimed below the base of the sternum, according to procedures that have previously described practices for obtaining consistent temperature readings , 27. BecStatistical analyses were performed as indicated in figure legends using GraphPad Prism 6.0 software.V. vulnificus strain HG0902 succumbed to infection within 24 hpi  was 17 hpi (S.D. ± 8 hpi) with a range of 8 to 40 hpi. In the course of these experiments, the VST was also measured  reading during the experiment was 21.1 °C and the maximum VST (VSTmax) was 33.7 °C for a total measurement range of 12.6 °C.Data were first analyzed pooling results for all 0). This drop was measured in both infected and mock-treated animals  of tested animals exhibited a drop in VST relative to initial  and finally a period of temperature stability (16–48 hpi).Among  °C Fig. . Survivomin readings at 8–12 hpi  is the proportion of positive diagnostic calls that are, indeed, true positives. In this case, 14 mice reached VST ≤ 23.5 °C. 13 of these 14 mice were non-survivors. Therefore, the hypothermia endpoint has a PPV of 93%. Conversely, the negative predictive value (NPV) is the proportion of negative test calls that are true negatives outcomes. Twenty of the 26 mice that had temperatures above the cutoff were survivors, while 6 were non-survivors. Therefore, the hypothermia endpoint VST ≤ 23.5 °C has a NPV of 77%. This analysis supports the conclusion that reduction in VST is predictive of subsequent death.The survival data were next analyzed comparing temperature-predicted outcomes to actual outcomes. To assess the efficacy of hypothermia as an experimental endpoint when comparing different infecting bacterial strains, time-to-death and time-to-VST ≤ 23.5 °C were compared for each infecting strain in this study Fig. . In the 2, Table 2 analysis revealed no significant differences between binary survival outcomes when the hypothermia plus actual death endpoint was applied  was analyzed by chi-square test χ, Table 2ied Fig. , Table 2ied Fig. , but eveied Fig. , Table 2p < 0.05. Interestingly, there were no statistical differences between endpoint curves when analyzed by log-rank test  determination. These studies need not take experimental kinetics into account, because LD50 is determined by the number of survivors and non-survivors at a given pathogen dose. In the current study, 68% of mice that spontaneously died could have been euthanized on average 10 h prior to death using the defined VST endpoint. At 95% sensitivity, application of this endpoint did not compromise experimental efficacy. Thus, applying an endpoint of VST ≤ 23.5 °C during V. vulnificus infection has potential to considerably reduce animal suffering in studies where binary data is desired. Our finding that binary live vs. dead outcomes are predictable by hypothermia suggests that the VST alternative endpoint is appropriate for LD50 determination and should be applied to LD50 studies to reduce suffering of animals in these experiments.In many cases, differences in pathogen virulence are best defined by median lethal dose  use smaller numbers of mice in a survival curve analysis without the option of a humane endpoint, a decision that will depend largely on the specific experimental goals of the research group in question.Interestingly, these results leave researchers to determine whether it is more ethical to: (A) use larger numbers of mice and perform LDV. vulnificus infections had not previously been examined. It was here determined that surviving and non-surviving V. vulnificus infected mice demonstrate distinct temperature responses. Non-surviving infected mice have significantly lower VSTmin as compared to surviving mice. From these data, a temperature endpoint of 23.5 °C was empirically determined and retrospectively applied to the survival results. When used as an experimental endpoint, VST ≤ 23.5 °C exhibited sensitivity of 68% and specificity of 95%. The temperature cutoff of 23.5 °C demonstrates 93% PPV and 77% NPV. Given the outcomes of this study, a hypothermia-based humane endpoint can be applied to LD50 studies without concern of reducing the efficacy of the experiment; binary (live vs. dead) survival outcomes are almost always numerically identical and in all cases statistically indistinguishable from survival data using death as an endpoint (Fig. While hypothermia has been demonstrated as a key marker of death due to infectious disease, its relationship to survival of int Fig. , Table 2By its very nature, an alternative endpoint aims to predict death early such that animal research subjects can be euthanized prior to death. VST ≤ 23.5 °C predicts non-survival outcomes an average of 10 h in advance of the event. This allows for a considerable reduction in unnecessary mouse infection hours. Nonetheless, given that a hypothermia endpoint alters experimental kinetics, these kinetic changes suggest that VST endpoint should not be applied to survival curve experiments.


Incoming Chief Editor Philippe Sansonetti shares his vision of the role of the journal in the burgeoning areas of molecular medicine. Following after clinical anatomy and experimental medicine, molecular medicine is medicine's third revolution in the last two centuries. Molecular medicine is a complex discipline that aims at deciphering the structural, molecular, and cellular pathways and signals underlying disease processes, identifying the possible genetic defects causing these dysfunctions, and thus to design molecular tools and interventions to diagnose, prevent and treat these diseases. Molecular medicine is in essence a multidisciplinary exercise that requires integrating physics, chemistry, biology, and increasingly bioinformatics and big data management.EMBO Molecular Medicine, even if it took a few years before we could celebrate its birth in 2009 at another memorable kickoff workshop, this time at EMBL.EMBO could, of course, not ignore the molecular dimension of medicine. I attended the first EMBO sectorial meeting for molecular medicine organized by Frank Gannon in Dubrovnik in 2003. It was a revealing event, where cancer biologists, microbiologists and infection biologists, cardiovascular and kidney diseases experts, neuroscientists, gastroenterologists, hepatologists, endocrinologists, and experts in metabolic diseases realized that they shared a common language that transcended the various disciplines: the language of molecules, signals, and their regulation. At this meeting, I discovered the details of ubiquitination from talks on the genetic and molecular bases of the von Hippel‐Lindau syndrome and Fanconi anemia—since, covalent modification by small moieties like ubiquitin has become a dominant theme in infection signaling. This meeting laid the foundation for EMBO Molecular Medicine in the field. She did a great job, together with two outstanding editors, Céline Carret and Roberto Buccione, to set the demanding standards that have secured the strong image of scientific quality and editing efficiency and transparency that characterize EMBO Molecular Medicine, and place it well in line with the other three EMBO Press journals.This is also an opportunity to thank and congratulate Stefanie Dimmeler, my predecessor as Chief Editor, for firmly establishing the recognized position of As new Chief Editor, it will be my duty to pursue and buttress this successful editorial policy. I am strongly attached to this journal and its aim to promote molecular medicine, irrespective of its geographic source or subdiscipline, as long as contributions meet the EMBO standards of excellence.EMBO Molecular Medicine must continue to adapt to the fast‐changing landscape of molecular medicine. Geographically, the journal needs to have a more global outlook, and efforts will be made to strengthen the flux of American and Asian contributions. The journals' balance of disciplines also needs to be reequilibrated as some very active specialties remain under‐represented. This is the case, for example, with infection biology and metabolic diseases. Our senior editors will be mobilized to help restore visibility of these disciplines and we will collectively put the emphasis on commissioning high‐standard reviews with a true integrative dimension, particularly to set the stage for under‐represented disciplines.Having said this, We will also increase our efforts to make the journal more open and attractive to our community by encouraging submission of short “state‐of‐the‐art” commentaries and research papers by outstanding leaders, thereby permanently reevaluating the evolving boundaries of molecular medicine.EMBO Molecular Medicine is our journal, I am honored and thrilled to take this Chief‐Editorial position, but also strongly committed to work with the editorial advisory board to make it an even more attractive and successful forum.


Theoretical prediction of the mechanical properties of soft tissues usually relies on a top-down approach; that is analysis is gradually refined to observe smaller structures and properties until technical limits are reached. Computer-Assisted Molecular Modeling (CAMM) allows for the reversal of this approach and the performance of bottom-up modeling instead. The wealth of available sequences and structures provides an enormous database for computational efforts to predict structures, simulate docking and folding processes, simulate molecular interactions, and understand them in quantitative energetic terms. Tendons and ligaments can be considered an ideal arena due to their well defined and highly organized architecture which involves not only the main structural constituent, the collagen molecule, but also other important molecular “actors” such as proteoglycans and glycosaminoglycans. In this ideal arena each structure is well organized and recognizable, and using the molecular modeling tool it is possible to evaluate their mutual interactions and to characterize their mechanical function. Knowledge of these relationships can be useful in understanding connective tissue performance as a result of the cooperation and mutual interaction between different biological structures at the nanoscale.


Single genome sequencing was used to amplify HIV RNA and DNA, which was present in all tumors. Independent HIV tumor clades in phylogenies differentiated KS1 and KS2 from KS3, whose sequences were interrelated by both phylogeny and selection. HIV compartmentalization was confirmed in KS1 and KS2 tumors; however, in KS3, no compartmentalization was observed among sampled tissues. While the sample size is small, the HIV evolutionary patterns observed in all patients suggest an interplay between tumor cells and HIV-infected cells which provides a selective advantage and could promote KS progression.Kaposi's sarcoma (KS) in HIV-infected individuals can have a wide range of clinical outcomes, from indolent skin tumors to a life-threatening visceral cancer. KS tumors contain endothelial-related cells and inflammatory cells that may be HIV-infected. In this study we tested if HIV evolutionary patterns distinguish KS tumor relatedness and progression. Multisite autopsies from participants who died from HIV-AIDS with KS prior to the availability of antiretroviral therapy were identified at the AIDS and Cancer Specimen Resource (ACSR). Two patients (KS1 and KS2) died predominantly from non-KS-associated disease and KS3 died due to aggressive and metastatic KS within one month of diagnosis. Skin and visceral tumor and nontumor autopsy tissues were obtained ( Early in the HIV epidemic, KS was so frequently diagnosed that it was considered an AIDS-defining illness [+ patients had a 40% lifetime risk of developing KS [+ patients [+ T-cell counts, low viral loads, and reduced risk of opportunistic infections [+ patients with disseminated disease [Prior to the human immunodeficiency virus (HIV) epidemic, Kaposi's sarcoma (KS) was considered a very rare disease, found primarily in Mediterranean countries and in organ transplant recipients . Althoug illness –5. Prioroping KS . Althougoping KS –9, the soping KS , 11. Furpatients  despite fections –16. KS ifections –19; howe disease .KS lesions can be solitary, localized, or disseminated, but the relationship among tumors in a single individual is largely unknown. While many KS lesions are cutaneous and indolent, tumors can occur in the oral cavity, lymph nodes, and viscera, which can result in fatal complications due to interference with normal body function. The growth of lesions can be very slow or explosively fast. The KS defining cell-type is a spindle cell (SC) , 22 that post mortem from the AIDS and Cancer Specimen Resource (ACSR) from three HIV+/KS+ patients who died from aggressive AIDS, and we generated HIV env-nef sequences using single genome sequencing (SGS) to characterize viral evolutionary patterns.HIV has the potential to infect and evolve in many anatomical tissues, including tumors –27. Prevhttp://acsr.ucsf.edu/). Tissues were classified as tumor or nontumor by histological examination. The ACSR is a National Cancer Institute-funded tissue-banking program that obtains tissues from patients after appropriate consent and releases tissues to investigators following approval of the proposed study and deidentification of samples and clinical histories. The ACSR is recognized by the Office of Biorepositories and Biospecimen Research at the National Institutes of Health as being HIPAA compliant and in accordance with the ethical standards of the Declaration of Helsinki. Additionally, all material was obtained under approval from the University of California at San Francisco Committee on Human Research.The three male patients included in this study were diagnosed with HIV infection and died with advanced AIDS without receiving cART . 100 mg μL final elutions using RNase-free water during the RNA isolation. The 100 μL final volume of RNA generated was concentrated using RNeasyMinElute Cleanup Kit (Qiagen #74204). RNA and DNA extractions, cDNA synthesis, and first-round PCR setup were conducted in a restricted-access, amplicon-free room with separate air handling and laboratory equipment where no amplified PCR products or recombinant cloned plasmids were allowed, and work surfaces and equipment were thoroughly cleaned before and after use with Eliminase® . cDNA was synthesized using SuperScript® III First-Strand Synthesis System (Invitrogen Life Technologies #18080-051) using the provided Oligo(dT)20 primer according to manufacturer's recommendations. RNA was incubated at 65°C for 5 minutes with deoxynucleoside triphosphates (0.5 mM) and 5 μM Oligo(dT)20 and then cooled quickly to 4°C. First-Strand cDNA Synthesis was performed in a 40 μL reaction volume containing 1x reverse transcription buffer , 5 mM MgCl2, 10 mM dithiothreitol, 2 U/μL of RNase-OUT™ (RNase inhibitor), and 10 U/μL SuperScript III RT. The reaction was heated to 50°C for 50 minutes, followed by 85°C for 5 minutes. The reaction was cooled to 37°C and 0.1 U/μL of E. coli RNase H was added, followed by a 20-minute incubation. cDNA was stored at −20°C.Total RNA and genomic DNA were isolated separately from each tissue section (30–50 ng) using AllPrep DNA/RNA Mini Kit (Qiagen #80204). Tissues were homogenized just prior to extraction in Buffer RLT Plus (lysis buffer) using a TissueRupter rotor-stator homogenizer (Qiagen #9001271) with a fresh sterile disposable probe (Qiagen #990890) for each sample. Manufacturer's guidelines were followed, with the exception of two 50  env and nef sequences [μL reactions containing 1x Platinum® Blue PCR SuperMix (Invitrogen Life Technologies) and 0.2 μM of each primer: EF2, 5′-ACAGTCTATTATGGGGTRCC-3′ and NR1, 5′-AGCTCCCAGGCTCAGATCT-3′  with the following cycling parameters: initial denaturation 95°C for 5 minutes, followed by 35 cycles of denaturing at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 4 minutes, with a final extension time of 10 minutes at 72°C. A second round of gp120 PCR consisted of 1 μL of the first-round PCR added to a 24 μL second-round reaction consisting of 1x Platinum Blue PCR SuperMix (Invitrogen Life Technologies) and 0.2 μM of each primer: EF3, 5′ CATAATGTTTGGGCCACACA-3′ and ER2, 5′-CACCACTCTTCTYTTTGCC-3′  with the following cycling parameters: initial denaturation 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 2 minutes, with a final extension time of 10 minutes at 72°C. This second-round PCR generated a 1.3 Kb product, nearly covering the entire env gp120 gene. Second-round env gp120 PCR products were visualized on 1% agarose gels stained with ethidium bromide, and reactions containing a single 1.3 Kb product were considered positive and selected for sequencing. Subsequently, the first-round reactions that corresponded to positive second-round gp120 PCRs were then used to amplify the nef gene sequence. Second-round nef PCR consisted of 1 μL of the first-round PCR added to a 24 μL second-round reaction consisting of 1x Platinum Blue PCR SuperMix (Invitrogen Life Technologies) and 0.2 μM of each primer: NF1, 5′-TTAGGCAGGGATAYTCACC-3′ and NR2, 5′-ATCTGAGGGCTCGCCACT-3′  with the following cycling parameters: initial denaturation 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 2 minutes, with a final extension at 72°C for 10 minutes. Second-round nef PCRs were visualized on 1% agarose gels stained with ethidium bromide, and reactions containing single 1.1 Kb products were considered positive and selected for sequencing. The primers were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) and by observing regions of conservation in alignments of published HIV subtype B sequences downloaded from Los Alamos HIV Sequence Database (http://www.hiv.lanl.gov/). Sequencing was performed on an Applied Biosystems 3730xl DNA Analyzer (Life Technologies) at the University of Florida Interdisciplinary Center for Biotechnology Research (UF ICBR). All sequences were assembled and aligned with the Geneious R7 software package (Biomatters http://www.geneious.com/) followed by a manual optimization of regions containing insertions and deletions. Sequence data has been submitted to GenBank (Accession numbers KU709129–KU709831).A modified single genome sequencing protocol was used to amplify linked HIVequences . cDNA anEnv and nef alignments were generated using ClustalW [ env V1, V2, and V4 domains were removed. An initial maximum-likelihood phylogeny was inferred using sequences from all patients for both genes to ensure no cross-contamination. Pairwise distance analysis within and between RNA and DNA tissue sequence populations over time was assessed in MEGA5 [http://datamonkey.org/), were used to identify amino acid sites under selection in sequences derived from different tissues for each individual. Sites with posterior probability >0.9 of an increased (diversifying) or decreased (purifying) rate of nonsynonymous relative to synonymous substitutions were considered to have experienced a significant level of selective pressure. Selected sites were numbered according to the molecular HIV clone HXB2 (GenBank Accession #AF358141).ClustalW  in MEGA5ClustalW  with furin MEGA5  using thin MEGA5 . Statistin MEGA5  was usedin MEGA5 . The fas3 at death and were diagnosed with KS only months prior to death  four months prior to death that was treated with chemotherapy. BCBL may be related to primary effusion lymphoma which is typically caused by HHV8, the Kaposi sarcoma herpes virus. KS3 had a nine-year history of HIV infection with reoccurring upper respiratory tract infections. He was diagnosed with KS one month prior to death. His KS lesions became rapidly disseminated on the skin and internally, which lead directly to his death due to respiratory distress.All patients (KS1–KS3) had CD4 counts <50 cells/mmto death . KS1 livViral sequences were obtained from RNA in 13/14 tissues and from DNA in 12/14 tissues . A preli env and nef sequence populations for all three patients. In the KS1 env phylogeny, four distinct and well-supported clades (A–D) were apparent that contained the majority of tumor-derived sequences . Similarly, all skin tumor-derived sequences except one clustered together in a distinct and well-supported clade in KS2 for both env and nef (A) (n = 9). Branches in the nontumor clade showed considerable structure in both genes, consistent with a continuously diversifying virus population. In the nef phylogeny, two distinct clades were evident within the mixed clade, which was not present in the env tree. In contrast, for KS3, all tumor- and nontumor-derived sequences were largely interspersed in both env and nef phylogenies (ML phylogenies were inferred forequences . The rel nef (A) . Small blogenies . Three wp < 0.001) (KS1 lymph node tumor nef versus nontumor nef sequences). Viral populations were significantly compartmentalized within each of the two tumors and normal tissues for both env and nef in KS1 and KS2 (p < 0.001). On the other hand, gene flow among the three sites was found in both genes in KS3 . Viral populations from combined tumor locations were again significantly compartmentalized compared with nontumor populations in env and nef in KS1 and KS2, as well as in nef for KS3 (p < 0.001).When pairwise distances were calculated for sequences derived from each tumor and nontumor tissue, only one significant difference was observed . In nef, a significantly higher number of negatively selected codons over positively selected codons were observed in all patients (p < 0.01). Codons under selection were tissue-specific in both KS1 and KS2, with only one being shared between two tissues in KS2. In contrast, in KS3 env sequence populations, all shared positively and negatively selected codons in tumor tissues were also identified within nontumor sequences; similarly, in KS3 nef sequence populations, all shared selected codons from tumors were identified in nontumor tissues except codon positions 31 and 67.The proportion of codons under selection in env and nef sequences from tumor and nontumor anatomical sites from three subjects who died with KS. Medical records indicated that subjects KS1 and KS2 had a slower KS progression than subject KS3; however, all patients had visceral KS, indicating an advanced cancer at death from AIDS. HIV RNA and/or DNA was amplified from all tumors in the study, demonstrating that, similar to a previous study [In this study we examined if HIV evolutionary patterns could differentiate metastatic KS in patients untreated with cART. We analyzed HIVus study , cancer HIV from tumor sites in KS1 and KS2 was compartmentalized relative to the nontumor virus, with limited gene flow between compartments; however, subpopulations of virus were also present within tumors. Additionally, codon selection analysis demonstrated that, at the protein level, KS1 and KS2 had sequence populations evolving under independent selective pressures in different tumor/nontumor sites. In contrast, the patient who died from highly aggressive KS (KS3) showed remarkably different patterns. Here the virus in tumor and nontumor tissue was completely interspersed with a non-tissue-specific viral population structure. These findings could result from a number of scenarios, including the following: (1) subpopulations of virus in tumor could result from recent viral migration from another, unsampled anatomical location; (2) the viral population in the tumors is evolving independently due to a physical barrier restricting gene flow; (3) the viral populations in the tumors are evolving in response to local selective pressures; (4) viral sequences in one or more sites could represent archival virus that was reactivated because of the cancer growth. The first scenario is untestable here; however, we are conducting additional studies with more tumor and nontumor tissues from other KS patients, including KS derived from patients on cART with no detectable plasma viral load. While the second scenario is possible, unlike the brain in which viral flow is impeded by the blood-brain barrier, KS tumors are highly vascularized which would in theory provide ample opportunity for migration. Furthermore, a small number of tumor viruses were found in nontumor tissues, also suggesting a lack of physical barrier. In considering the third and fourth scenario, we did find that codon positions were under varied selective pressures in the tumors from KS1 and KS2 when compared to nontumor tissues (as expected from the phylogeny). For KS3, most, but not all, selected codons were shared among tissues; however, this patient had widely metastatic KS at the time of death. Therefore, it is possible that tumor-compartmentalized HIV populations could have existed in this patient prior to the rapid outgrowth and spread of the cancer, perhaps represented by one of the distinct subpopulations. c-fes oncogene, which could contribute to clonal expansion [+ from the tumor to nontumor sites [Reactivation and clonal expansion of viral populations could be consistent with the large clades of identical or nearly identical tumor-associated virus in KS1 and KS2 phylogenies and with the separation of DNA and RNA derived sequences into different clades within tumors of KS1. This hypothesis could be investigated further by identifying the infected cells types within each tumor. Furthermore, identifying the insertion sites within the host genome in these tumors could help understand if HIV-infected cells in tumors are physically migrating to other tumor sites. A recent HIV integration site study showed that one mechanism of HIV persistence within T-cells is related to clonal expansion . Interesxpansion –38. Thisor sites .The patients studied here were all cART-naïve; however, KS remains a substantial comorbidity even for patients undergoing cART. For some patients, rapid progression of KS occurs under cART even with an undetectable viral load . These cKaposi's sarcoma (KS) is a major cancer that still occurs in HIV positive patients despite immune-restorative combined antiretroviral therapy (cART). In this study we isolated HIV from tumor and nontumor tissue sites from patients who died with KS which had metastasized to the viscera (KS1–KS3). KS1 and KS2 died from other AIDS-associated complications, whereas KS3 died directly due to a highly aggressive KS that metastasized to lungs and caused death within 1 month of initial KS diagnosis. We compared the HIV sequences among patient's tissue sites and found that KS1 and KS2 had unique HIV in tumors compared to nontumor sites, whereas HIV was freely migrating among all tissues in KS3. While the study is based on a very small sample size, the results suggest that an HIV-associated mechanism may promote the metastatic process in aggressive KS. It is also important to note that it is unclear how the other AIDS-associated cancers may have impacted the study . Continued studies of the evolutionary patterns within and among KS tumors could help in understanding the mechanisms of metastasis as well as describing an additional HIV reservoir during cART. Evolutionary studies of HIV in tumor tissues are sorely lacking, and further investigation could provide additional clarity into the mechanisms of HIV-associated cancers.


Chronic kidney disease (CKD) is a major health problem worldwide. In Europe it is estimated that ~16% of the general population has CKD stages 1–4 and in US the prevalence is similar at ~13%. CKD is associated with increased risk of cardiovascular disease and mortality. It is therefore important to further our understanding regarding the molecular determinants of CKD and find new ways to manipulate them for therapeutic benefit. A large proportion of CKD is due to diabetic nephropathy (DN), one of the microvascular complications of diabetes. In the Western world, DN is the leading cause of end-stage renal disease and kidney replacement therapy , an immense burden on healthcare costs. It is estimated that 40–45% of patients with type I diabetes and 30% of patients with type II diabetes have nephropathy.Multiple molecular mechanisms of DN progression have been described in recent years; however, mechanism-derived treatments for DN are still lacking. This is partly due to poor understanding of the detailed molecular mechanisms underlying diabetic complications. The standard of care is to control glycaemia but most of the time this does not stop the occurrence of the nephropathy.There is therefore an increasing need to better understand molecular mechanisms of progression in DN and to develop novel treatments that target specifically these mechanisms and are able to slow progression of the underlying CKD in DN.This special issue is trying to address these goals by publishing both articles that describe novel molecular pathways implicated in DN and novel treatment ideas.We do hope that the papers here collected may offer the readers an improved knowledge on innovative and original advances in DN pathogenesis and management.Sebastian OlteanRichard CowardMassimo CollinoHans Baelde


Knobloch Syndrome (KS) is a rare congenital syndrome characterized by occipital skull defects and vitreoretinal degeneration. Retinal detachment (RD) often occurs at the end of the first decade of life or later. Aside from occipital skull defects, central nervous system abnormalities are uncommon.COL18A1, diagnostic of KS. MRI of both siblings demonstrated polymicrogyria but did not show occipital defects.We report on two siblings with KS. The first, a seven month old male, presented with nystagmus and was found to have a serous RD and a tessellated retinal appearance. His sister had a history of multiple visual abnormalities and had a similar retinal appearance although no signs of RD, but retina staphylomas. Genetic testing performed on both siblings showed a mutation in Although several families with KS have been described previously, our case is noteworthy for several reasons. The RD observed in our first patient occurred at an early age, and we find evidence of only one patient with KS who had an RD identified at an earlier age. The findings of polymicrogyria are not characteristic of KS, and we found only a few previous reports of this association. Additionally, we review potential treatment options for this condition. Knobloch Syndrome (KS) is a rare autosomal recessive syndrome first described in 1971 characterized by vitreoretinal degeneration and occipital skull abnormalities [Midline occipital defects, namely bone defects, encephalocele, or aplasia cutis congenita, are characteristic findings. Other central nervous system findings are overall rare and not considered to be stereotypic features of KS. Caglayan et al. review seven cases of patients with KS associated with other central nervous system findings including pachygyria, polymicrogyria and cerebellar atrophy among other findings . DevelopCOL18A1, which encodes for collagen type XVIII α-1 chain. It is ubiquitously expressed in vascular and epithelial basement membranes and has multiple functions in ocular and neurologic development including maintenance of the basement membrane, cell proliferation, and angiogenesis [The causative gene in KS has been identified as ogenesis .Herein, we describe two siblings with KS associated with polymicrogyria, an anomaly sporadically associated with KS , 6. PolyA 7-month-old Hispanic male was referred for nystagmus. He was born at term and exhibited normal growth but had motor and social developmental delay. Family history was significant for an older sister (Case 2) with visual abnormalities. His parents and two older brothers did not have any clinically significant visual problems. On examination, the patient fixed and followed with the left eye (OS) but not with the right eye (OD). Cycloplegic refraction (CR) was found to be −2.50 + 3.50 × 090° OD and −7.00 + 3.50 × 090° OS. Anterior segment examination was unremarkable for both eyes (OU). Funduscopic examination OD showed a tilted optic nerve with trace pallor and a large posterior RD involving the macula with surrounding demarcation lines and a subretinal fibrotic band Fig. . The remCOL18A1 gene (NM_130445.3:c.2970_2971delAGinsC) associated with KS. Magnetic resonance imaging (MRI) of the brain demonstrated findings consistent with polymicrogyria but no evidence of an encephalocele  production. Interestingly, treating the mutants with the angiotensin II type 1 receptor antagonist losartan, a conventional hypertensive medication, has been shown to attenuate mitochondrial ROS production, improve mitochondrial morphology and restore function, suggesting a viable avenue for further investigation. Considerable research interest in the ocular renin-angiotensin system and its role in disease may help guide future treatment options for patients with KS [While treatment for KS is often supportive, recent advancements in our understanding of the pathophysiology of the disease come from studies in osophila . Mutatio with KS .Further investigation is necessary to enhance our understanding of the pathophysiology of KS so that we may offer improved medical and surgical treatments for our patients.