Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons.

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. In contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations.

Developing antimicrobial calcium alginate fibres from neem and papaya leaves extract.

OBJECTIVE: The aim of this study was to evaluate an alternative approach to developing antimicrobial alginate fibres using plant extracts, such as those from neem and papaya leaves. METHOD: Aqueous leaves extract from neem, papaya and their hybrid combinations were used as solvent to develop sodium alginate fibres. Sodium alginate polymer containing these extracts was extruded in a calcium chloride (CaCl2) bath to develop neem (NE), papaya (PE) and their hybrid combinations-based calcium alginate fibres (H-1 to H-5). The surface morphology, spectra, liquid absorption, tensile strength and antimicrobial activity of these developed fibres were measured. RESULTS: NE fibre showed greater tensile strength than PE. The liquid absorption property of all developed fibres decreased, but showed antibacterial properties against Staphylococcus aureus and reduced bacterial growth up to 85% when compared with pure calcium alginate fibre. CONCLUSION: Neem, papaya and hybrid extract-based calcium alginate fibres have the potential to be used as wound dressings.

In situ development and application of natural coatings on non-absorbable sutures to reduce incision site infections.

OBJECTIVE: The purpose of the study was the development of a suture line that has antibacterial properties and reduces the chance of wound infection thus facilitating the healing process. METHOD: Hydrolysed chitosan, turmeric powder and clove oil were used in different proportions to formulate antimicrobial coating for the polyethylene terephthalate (PET) and polyamide (Nylon 6) threads. The threads were coated using a lab-scale yarn sizing machine. Tensile, and knot strength of the coated sutures were measured. As was the antimicrobial action of Staphylococcus aureus strain ATCC29213. RESULTS: The results show that coatings have slightly improved the tensile and knot strength properties of these sutures. The coated sutures also have satisfactory microbial inhibition against Staphylococcus aureus. CONCLUSION: The coating slightly improved the tensile strength of the sutures. However, the knot is the weakest part of the suture strand. All the formulations of the coating have shown satisfactory antimicrobial activity against Gram-positive Staphylococcus aureus bacteria. We conclude that application of natural coatings on non-absorbable sutures can be useful to reduce the incisions and wound site infections.

Novel materials for moist wound management: alginate-psyllium hybrid fibres.

OBJECTIVE: To assess the feasibility of producing high absorbing, high gelling hybrid fibres made from alginate and psyllium. METHOD: Three methods of psyllium extraction were attempted: the hot, cold and hydrolysed method. The extracted psyllium was sieved before addition to the alginate dope. To assess feasibility, each dope was in turn extruded on a bench-top extruder and then on a pilot-size wet extruder. Calcium chloride solution was used as the coagulation medium for all the extrusions. The produced fibres were subsequently assessed for their linear density, tensile strength, absorption characteristics and swelling behaviour. Optical microscopy was also used to illustrate hybrid fibre swelling behaviour under different conditions RESULTS: This feasibility study has shown that hybrid fibres can be produced when using different methods of psyllium extraction, proving the feasibility of the proposed methodology. However, based on physical and functional assessment of the fibres, it was found that the cold water extraction method leads to better hybrid fibres with superior tensile properties, liquid absorption and swelling, which would render them most suitable for heavily exudating wounds. CONCLUSION: The cold water route is found to be the most effective way of producing polysaccharide-based hybrid fibres. This method is also the least expensive, both in terms of room temperature preparations and functional effectiveness, using only 0.75% psyllium. This fibre is therefore recommended for wound dressing applications. DECLARATION OF INTEREST: There were no external sources of funding for this study. The authors have no conflicts of interest to declare.

Evaluation of clinical efficacy of streptokinase by comparison with the thrombolytic agent on animal model.

Cardiovascular disorders, including acute myocardial infarction (AMI), often lead to blood clot formation, impacting blood circulation. Streptokinase, a cost-effective and widely available thrombolytic agent, is crucial in treating thrombosis. This study aimed to produce streptokinase from Streptococcus pyogenes EBL-48 and compare its efficacy with heparin in an animal model. We evaluated the clot-lysing effectiveness of streptokinase produced from Streptococcus pyogenes EBL-48, emphasizing its low cost and ease of production. Streptokinase was produced using pre-optimized fermentation media and purified through ion exchange and gel-filtration chromatography. In vivo analysis involved inducing clots in a trial animal model using ferric chloride, comparing streptokinase with heparin. Ultrasonography assessed the clot-lysing activity of streptokinase. Streptokinase (47 kDa) effectively lysed clots, proving its low cost, easy production, and minimal adverse effects. Ultrasonography confirmed its fibrinolytic efficacy. These findings highlight potential as an affordable and easily produced thrombolytic agent, particularly relevant in resource-limited settings. Streptokinase efficacy and minimal adverse effects make it a promising option for thrombolytic therapy, especially in economically constrained regions. Future studies could optimize production techniques, explore different strains, and conduct clinical trials for human validation. Comparative studies with other thrombolytic agents would enhance understanding of their advantages and limitations.

Unveiling intrinsic material and extrinsic pinning dimensionality in superconductors: Why Fe(Se,Te) is able to mimic YBCO.

Discovery of iron-based superconductors paved the way to a competitor of high-temperature superconductors, easier to produce, better performing in high fields, and promising to be less expensive. Critical parameters are investigated by resistivity measurements as a function of temperature, field, and angle R(T,H,θ). This work presents a deep analysis of H-θ phase diagram of PLD-processed Fe(Se,Te) superconducting films, thus revealing material and pinning anisotropy at once. By selecting different thresholds along the R(T,H,θ) curves, all possible regimes emerge. Surprisingly, anisotropy arises moving from the upper critical field toward the irreversibility line: gradually a non-monotonous transition from 3D to 2D, and backward to 3D occurs. Although Fe(Se,Te) appears as a 3D superconductor, its anisotropic pinning landscape shows up similarities with an intrinsic layered superconductor and Fe(Se,Te) definitively mimics YBCO. We propose a general method to disentangle, in any other superconductor, material dimensionality and pinning anisotropy that are key constraints for applications.

A Novel Algorithm for Improving the Prehospital Diagnostic Accuracy of ST-Segment Elevation Myocardial Infarction.

INTRODUCTION: Early detection of ST-segment elevation myocardial infarction (STEMI) on the prehospital electrocardiogram (ECG) improves patient outcomes. Current software algorithms optimize sensitivity but have a high false-positive rate. The authors propose an algorithm to improve the specificity of STEMI diagnosis in the prehospital setting. METHODS: A dataset of prehospital ECGs with verified outcomes was used to validate an algorithm to identify true and false-positive software interpretations of STEMI. Four criteria implicated in prior research to differentiate STEMI true positives were applied: heart rate <130, QRS <100, verification of ST-segment elevation, and absence of artifact. The test characteristics were calculated and regression analysis was used to examine the association between the number of criteria included and test characteristics. RESULTS: There were 44,611 cases available. Of these, 1,193 were identified as STEMI by the software interpretation. Applying all four criteria had the highest positive likelihood ratio of 353 (95% CI, 201-595) and specificity of 99.96% (95% CI, 99.93-99.98), but the lowest sensitivity (14%; 95% CI, 11-17) and worst negative likelihood ratio (0.86; 95% CI, 0.84-0.89). There was a strong correlation between increased positive likelihood ratio (r2 = 0.90) and specificity (r2 = 0.85) with increasing number of criteria. CONCLUSIONS: Prehospital ECGs with a high probability of true STEMI can be accurately identified using these four criteria: heart rate <130, QRS <100, verification of ST-segment elevation, and absence of artifact. Applying these criteria to prehospital ECGs with software interpretations of STEMI could decrease false-positive field activations, while also reducing the need to rely on transmission for physician over-read. This can have significant clinical and quality implications for Emergency Medical Services (EMS) systems.

Education and training in NOTES: a systematic review.

INTRODUCTION: Since the first published work on natural orifice translumenal endoscopic surgery (NOTES) a decade ago, progress has been made in the domain of education and training, although questions posed by the original White Paper remain. This article aims to review the current status of education and training in NOTES. METHODS: A review of the literature was conducted to evaluate the following: (1) What are the current training methods/modalities used for NOTES; what is the level of evidence to support their use? (2) How has NOTES clinical training been quantified; what is the evidence relating to performance of different NOTES operators? (3) What clinical NOTES training programs have been established and what are the wider training needs? RESULTS: A total of 25 studies were included: 11 nonanimal studies, 8 animal studies, and 6 descriptions of education programs. Several animal and simulator models demonstrated construct validity, but no study showed human predictive validity. Logarithmic learning curves in animal models demonstrate 10 to 15 cases achieving a proficiency level. Current trends are that gastroenterologists prefer it for diagnostic and basic procedures, whereas surgeons prefer it for complex therapeutic cases. CONCLUSION: The development of a new specialty is intriguing but currently unviable. Training programs have been initiated, but information is limited; the common theme is surgeons receiving endoscopic training. Despite the research done, our knowledge of training and educating in NOTES procedures is limited, preventing a meta-analysis or formal review from being performed. Further research is needed to integrate NOTES into routine clinical procedure.

Agro-morphological characterization of Pakistani maize accessions using qualitative and quantitative traits.

Agro-morphological characterizations offer robust and vigorous means for the precise characterization of germplasm to be used in breeding programs. Here, agro-morphological features-based analyses were considered to figure out the genetic variability within 99 maize accessions and five (5) check varieties including Azam, W. Islamabad, Haq Nawaz, Ev-5 and Evr197. A total of 28 important agro-morphological traits were accounted in the field trails at Hazara University Mansehra Pakistan, during spring 2015. The maximum variation was detected in grain weight per cob (53.43), followed by number of kernels per row (38.64) and flag leaf area (cm2), (38.09%). Cluster analysis divided the 99 maize accession with five check varieties of maize accessions into 7 clusters following the hierarchical clustering. Cluster II recorded 29 genotypes with maximum grain yield per cob, and highest flag leaf length, followed by cluster IV. Lowest grain yield per cob was noted for accessions of cluster I. Similarly, cluster VII consisted of accession with the longest cob length. Moreover, the investigations also revealed that the primary constituent among first five principal components with an eigen value about more than 0.98 in relation to 68.75% of the total variants. PCI accounted for 25.53%, PCII contributed 18.31%, and PCIII is 9.88% of the overall morphological variability were significant contributors were grain weight per cob, number of kernel per row, 1000 grain weight. The identification of a significant level of genetic diversity during the present investigation having implications for maize germplasm characterisation, conservation, and breeding programs aiming at developing improvement maize cultivars.

Characterization of wheat-Thinopyrum bessarabicum genetic stock for stripe rust and Karnal bunt resistance.

Utilization of modern breeding techniques for developing high yielding and uniform plant types ultimately narrowing the genetic makeup of most crops. Narrowed genetic makeup of these crops has made them vulnerable towards disease and insect epidemics. For sustainable crop production, genetic variability of these crops must be broadened against various biotic and abiotic stresses. One of the ways to widen genetic configuration of these crops is to identify novel additional sources of durable resistance. In this regard crops wild relatives are providing valuable sources of allelic diversity towards various biotic, abiotic stress tolerance and quality components. For incorporating novel variability from wild relative's wide hybridization technique has become a promising breeding method. For this purpose, wheat-Th. bessarabicum amphiploid, addition and translocation lines have been screened in field and screen house conditions to get novel sources of yellow rust and Karnal bunt resistant. Stripe rust screening under field conditions has revealed addition lines 4JJ and 6JJ as resistant to moderately resistant while addition lines 3JJ, 5JJ, 7JJ and translocation lines Tr-3, Tr-6 as moderately resistant wheat-Thinopyrum-bessarabicum genetic stock. Karnal bunt screening depicted addition lines 5JJ and 4JJ as highly resistant genetic stock. These genetic stocks may be used to introgression novel stripe rust and Karnal bunt resistance from the tertiary gene pool into susceptible wheat backgrounds.

Characterization of wheat-Thinopyrum bessarabicum genetic stock for stripe rust and Karnal bunt resistance / Caracterização do estoque genético de trigo-Thinopyrum bessarabicum para resistência de ferrugem e Karnal bunt

Abstract Utilization of modern breeding techniques for developing high yielding and uniform plant types ultimately narrowing the genetic makeup of most crops. Narrowed genetic makeup of these crops has made them vulnerable towards disease and insect epidemics. For sustainable crop production, genetic variability of these crops must be broadened against various biotic and abiotic stresses. One of the ways to widen genetic configuration of these crops is to identify novel additional sources of durable resistance. In this regard crops wild relatives are providing valuable sources of allelic diversity towards various biotic, abiotic stress tolerance and quality components. For incorporating novel variability from wild relative's wide hybridization technique has become a promising breeding method. For this purpose, wheat-Th. bessarabicum amphiploid, addition and translocation lines have been screened in field and screen house conditions to get novel sources of yellow rust and Karnal bunt resistant. Stripe rust screening under field conditions has revealed addition lines 4JJ and 6JJ as resistant to moderately resistant while addition lines 3JJ, 5JJ, 7JJ and translocation lines Tr-3, Tr-6 as moderately resistant wheat-Thinopyrum-bessarabicum genetic stock. Karnal bunt screening depicted addition lines 5JJ and 4JJ as highly resistant genetic stock. These genetic stocks may be used to introgression novel stripe rust and Karnal bunt resistance from the tertiary gene pool into susceptible wheat backgrounds.

Resumo A utilização de técnicas modernas de melhoramento para o desenvolvimento de tipos de plantas uniformes e de alto rendimento, em última análise, estreitando a composição genética da maioria das culturas. A composição genética restrita dessas plantações tornou-as vulneráveis a doenças e epidemias de insetos. Para uma produção agrícola sustentável, a variabilidade genética dessas culturas deve ser ampliada contra vários estresses bióticos e abióticos. Uma das maneiras de ampliar a configuração genética dessas culturas é identificar novas fontes adicionais de resistência durável. A esse respeito, os parentes selvagens das culturas estão fornecendo fontes valiosas de diversidade alélica para vários componentes de qualidade e tolerância ao estresse abiótico e biótico. Para incorporar a nova variabilidade da ampla técnica de hibridização de parente selvagem tornou-se um método de reprodução promissor. Para esse efeito, trigo-Th. As linhas anfiploides, de adição e translocação de bessarabicum foram selecionadas em condições de campo e de casa de tela para obter novas fontes de ferrugem amarela e resistência ao bunt de Karnal. A triagem de ferrugem em faixas em condições de campo revelou as linhas de adição 4JJ e 6JJ como resistentes a moderadamente resistentes, enquanto as linhas de adição 3JJ, 5JJ, 7JJ e as linhas de translocação Tr-3, Tr-6 como estoque genético de trigo-Thinopyrum bessarabicum moderadamente resistente. A triagem Karnal bunt descreveu as linhas de adição 5JJ e 4JJ como estoque genético altamente resistente. Esses estoques genéticos podem ser usados para introgressão da nova ferrugem e resistência ao bunt de Karnal do pool genético terciário em origens de trigo suscetíveis.

Characterization of wheat-Thinopyrum bessarabicum genetic stock for stripe rust and Karnal bunt resistance / Caracterização do estoque genético de trigo-Thinopyrum bessarabicum para resistência de ferrugem e Karnal bunt

Utilization of modern breeding techniques for developing high yielding and uniform plant types ultimately narrowing the genetic makeup of most crops. Narrowed genetic makeup of these crops has made them vulnerable towards disease and insect epidemics. For sustainable crop production, genetic variability of these crops must be broadened against various biotic and abiotic stresses. One of the ways to widen genetic configuration of these crops is to identify novel additional sources of durable resistance. In this regard crops wild relatives are providing valuable sources of allelic diversity towards various biotic, abiotic stress tolerance and quality components. For incorporating novel variability from wild relative’s wide hybridization technique has become a promising breeding method. For this purpose, wheat-Th. bessarabicum amphiploid, addition and translocation lines have been screened in field and screen house conditions to get novel sources of yellow rust and Karnal bunt resistant. Stripe rust screening under field conditions has revealed addition lines 4JJ and 6JJ as resistant to moderately resistant while addition lines 3JJ, 5JJ, 7JJ and translocation lines Tr-3, Tr-6 as moderately resistant wheat-Thinopyrum-bessarabicum genetic stock. Karnal bunt screening depicted addition lines 5JJ and 4JJ as highly resistant genetic stock. These genetic stocks may be used to introgression novel stripe rust and Karnal bunt resistance from the tertiary gene pool into susceptible wheat backgrounds.

A utilização de técnicas modernas de melhoramento para o desenvolvimento de tipos de plantas uniformes e de alto rendimento, em última análise, estreitando a composição genética da maioria das culturas. A composição genética restrita dessas plantações tornou-as vulneráveis a doenças e epidemias de insetos. Para uma produção agrícola sustentável, a variabilidade genética dessas culturas deve ser ampliada contra vários estresses bióticos e abióticos. Uma das maneiras de ampliar a configuração genética dessas culturas é identificar novas fontes adicionais de resistência durável. A esse respeito, os parentes selvagens das culturas estão fornecendo fontes valiosas de diversidade alélica para vários componentes de qualidade e tolerância ao estresse abiótico e biótico. Para incorporar a nova variabilidade da ampla técnica de hibridização de parente selvagem tornou-se um método de reprodução promissor. Para esse efeito, trigo-Th. As linhas anfiploides, de adição e translocação de bessarabicum foram selecionadas em condições de campo e de casa de tela para obter novas fontes de ferrugem amarela e resistência ao bunt de Karnal. A triagem de ferrugem em faixas em condições de campo revelou as linhas de adição 4JJ e 6JJ como resistentes a moderadamente resistentes, enquanto as linhas de adição 3JJ, 5JJ, 7JJ e as linhas de translocação Tr-3, Tr-6 como estoque genético de trigo-Thinopyrum bessarabicum moderadamente resistente. A triagem Karnal bunt descreveu as linhas de adição 5JJ e 4JJ como estoque genético altamente resistente. Esses estoques genéticos podem ser usados para introgressão da nova ferrugem e resistência ao bunt de Karnal do pool genético terciário em origens de trigo suscetíveis.

Characterization of wheat-Thinopyrum bessarabicum genetic stock for stripe rust and Karnal bunt resistance

Abstract Utilization of modern breeding techniques for developing high yielding and uniform plant types ultimately narrowing the genetic makeup of most crops. Narrowed genetic makeup of these crops has made them vulnerable towards disease and insect epidemics. For sustainable crop production, genetic variability of these crops must be broadened against various biotic and abiotic stresses. One of the ways to widen genetic configuration of these crops is to identify novel additional sources of durable resistance. In this regard crops wild relatives are providing valuable sources of allelic diversity towards various biotic, abiotic stress tolerance and quality components. For incorporating novel variability from wild relatives wide hybridization technique has become a promising breeding method. For this purpose, wheat-Th. bessarabicum amphiploid, addition and translocation lines have been screened in field and screen house conditions to get novel sources of yellow rust and Karnal bunt resistant. Stripe rust screening under field conditions has revealed addition lines 4JJ and 6JJ as resistant to moderately resistant while addition lines 3JJ, 5JJ, 7JJ and translocation lines Tr-3, Tr-6 as moderately resistant wheat-Thinopyrum-bessarabicum genetic stock. Karnal bunt screening depicted addition lines 5JJ and 4JJ as highly resistant genetic stock. These genetic stocks may be used to introgression novel stripe rust and Karnal bunt resistance from the tertiary gene pool into susceptible wheat backgrounds.

Resumo A utilização de técnicas modernas de melhoramento para o desenvolvimento de tipos de plantas uniformes e de alto rendimento, em última análise, estreitando a composição genética da maioria das culturas. A composição genética restrita dessas plantações tornou-as vulneráveis a doenças e epidemias de insetos. Para uma produção agrícola sustentável, a variabilidade genética dessas culturas deve ser ampliada contra vários estresses bióticos e abióticos. Uma das maneiras de ampliar a configuração genética dessas culturas é identificar novas fontes adicionais de resistência durável. A esse respeito, os parentes selvagens das culturas estão fornecendo fontes valiosas de diversidade alélica para vários componentes de qualidade e tolerância ao estresse abiótico e biótico. Para incorporar a nova variabilidade da ampla técnica de hibridização de parente selvagem tornou-se um método de reprodução promissor. Para esse efeito, trigo-Th. As linhas anfiploides, de adição e translocação de bessarabicum foram selecionadas em condições de campo e de casa de tela para obter novas fontes de ferrugem amarela e resistência ao bunt de Karnal. A triagem de ferrugem em faixas em condições de campo revelou as linhas de adição 4JJ e 6JJ como resistentes a moderadamente resistentes, enquanto as linhas de adição 3JJ, 5JJ, 7JJ e as linhas de translocação Tr-3, Tr-6 como estoque genético de trigo-Thinopyrum bessarabicum moderadamente resistente. A triagem Karnal bunt descreveu as linhas de adição 5JJ e 4JJ como estoque genético altamente resistente. Esses estoques genéticos podem ser usados para introgressão da nova ferrugem e resistência ao bunt de Karnal do pool genético terciário em origens de trigo suscetíveis.

Retinoid antagonism of NF-IL6: insight into the mechanism of antiproliferative effects of retinoids in Kaposi's sarcoma.

All-trans-retinoic acid (RA) is active in the treatment of Kaposi's sarcoma (KS), and retinoids inhibit KS cell growth in vitro. To understand the mechanism of retinoid action in KS, we studied the expression of autocrine growth factors of KS cells after RA treatment. We demonstrate that RA and its synthetic analogs inhibit the proliferation of KS cells by inhibiting the mRNA and protein levels of interleukin-6 (IL-6), an autocrine growth factor for KS cells. We further demonstrate that nuclear retinoid receptors (RA receptors [RARs] and retinoid X receptors [RXRs]) inhibit IL-6 promoter action by antagonizing the enhancer action of NF-IL6, a basic domain leucine zipper transcription factor belonging to the family of CAAT enhancer binding proteins. Furthermore, RARs and RXRs do not bind in vitro to an NF-IL6 binding site. However, the secondary folded structure of the DNA binding domain of RAR and RXR is obligatory for inhibiting NF-IL6 activity. Thus, NF-IL6 is a potential therapeutic target for the treatment of KS. Finally, using receptor-selective synthetic retinoids, we demonstrate that NF-IL6 antagonism and transactivation are separable functions of RAR alpha, thus indicating that synthetic retinoids with properties of NF-IL6 antagonism but lacking transactivation capabilities can be synthesized. Such retinoids might increase therapeutic potential in KS.

Vascular endothelial growth factor chimeric toxin is highly active against endothelial cells.

Angiogenesis is a critical step in a benign tumor's evolution toward malignancy and metastasis. Tumor cells acquire such a phenotype by their ability to secrete angiogenic factors such as vascular endothelial growth factor (VEGF). VEGF receptors (VEGFRs) flt-1/VEGFR-1 and Flk-1/ KDR/VEGFR-2 are restricted to activated endothelial cells, with the highest expression being in the tumor vasculature. The present study was undertaken to target the VEGFRs. Targeted toxins were developed by recombinant methods by fusing VEGF165 or VEGF121 to the diphtheria toxin (DT) translocation and enzymatic domain (DT390-VEGF165 or DT390-VEGF121). Both fusion proteins were found to be highly toxic to proliferating endothelial cells but not to vascular smooth muscle cells. The fusion protein is also active in Kaposi's sarcoma, a tumor type that expresses high levels of VEGFRs. These fusion proteins completely inhibit the basic fibroblast growth factor-induced growth of new blood vessels in the chick chorioallantoic membrane assay. Furthermore, the fusion toxin substantially retards the growth of Kaposi's sarcoma tumors in mice. Because nearly all tumors induce local angiogenesis with high VEGFR expression, VEGF-derived toxins may have wide application in cancer therapy.

Purification and properties of aspartate transcarbamylase from Mycobacterium smegmatis.

Aspartate transcarbamylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) has been purified from Mycobacterium smegmatis TMC 1546 using streptomycin sulphate precipitation, ammonium sulphate precipitation, DE-52 chromatography, second ammonium sulphate precipitation, Sephadex G-200 gel filtration, and aspartate-linked CNBr-activated Sepharose 4B affinity chromatography in successive order. The enzyme was purified 231.6-fold, and the preparation was found to be homogeneous on column chromatography and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 246,000 and was composed of two asymmetrical subunits. The kinetic and regulatory properties of aspartate transcarbamylase from M. smegmatis were also studied. The enzyme was found to be an allosteric in nature with carbamyl phosphate showing positive cooperativity and UMP exhibiting a negative cooperativity. CTP was found to be the most potent inhibitor among nucleotides. Phosphate acted as a non-competitive product inhibitor with respect to aspartate. Succinate and maleate exerted a competitive inhibition when aspartate was the variable substrate.

Interleukin 8 is an autocrine growth factor and a surrogate marker for Kaposi's sarcoma.

Kaposi's sarcoma (KS) is the most common tumor associated with HIV-1 infection. Here, we report the expression, regulation, and biological effect of interleukin (IL)-8 in KS. AIDS-KS cell lines expressed higher levels of IL-8 than either human umbilical vein endothelial cells (HUVECs), human aortic smooth muscle (AoSM) cells or fibroblast cells (T1). The inflammatory cytokine IL-1beta up-regulated IL-8 expression in a time- and concentration-dependent manner in KS cell lines. IL-8 antisense oligonucleotides specifically reduced IL-8 mRNA and protein levels and inhibited KS cell growth in a dose-dependent manner. In addition, supernatant from a KS cell line induced the growth of HUVECs and angiogenesis in chicken chorioallantoic membrane assays, both of which were inhibited by IL-8 neutralizing antibody. Serum levels of IL-8 were also elevated in KS cases compared with matched controls. Modulation of IL-8 may thus be of therapeutic value in this disease.

Selective killing of RPE with a vascular endothelial growth factor chimeric toxin.

PURPOSE: To determine the sensitivity of retinal pigment epithelial (RPE) cells to a vascular endothelial growth factor (VEGF) chimeric toxin. METHODS: A targeted toxin was developed using recombinant methods to fuse VEGF165 to the diphtheria toxin (DT) translocation and enzymatic domain (DT390-VEGF165). Human RPE cells, choroidal endothelial cells (CECs), and scleral fibroblasts were isolated, and a dose-response for DT390-VEGF165 was determined by measurement of cell proliferation and cell number. In parallel experiments, cultures were pretreated with transforming growth factor (TGF)-beta2. VEGF-receptor (VEGFR-1 and -2) expression was determined using reverse transcription-polymerase chain reaction and fluorescence-activated cell sorting, and affinity was measured using Scatchard analysis. RESULTS: RPE cells and CECs were similarly prone to killing by the VEGF-toxin, but scleral fibroblasts were unaffected. Pretreatment with TGF-beta2 selectively increased the sensitivity of RPE cells to the VEGF-toxin. RPE cells expressed both VEGFR-1 and -2 in vitro; however, the expression of VEGFR-1 was very low. Pretreatment with TGF-beta2 (10 ng/ml) was associated with increased expression of the VEGFR-1 in RPE cells and increased receptor affinity for VEGF detected by Scatchard analysis. CONCLUSIONS: Dose-dependent killing of RPE cells by the DT390-VEGF165 conjugate is selectively enhanced by pretreatment with TGF-beta2. This study provides further strong support for the presence of functional VEGFRs on human RPE cells, and demonstrates for the first time the ability to target a normal nonendothelial cell type through VEGFR expression.

Potentiation of cytotoxicity of Kaposi's sarcoma related to immunodeficiency syndrome (AIDS) by liposome-encapsulated doxorubicin.

Kaposi's sarcoma is an independent criterion for the diagnosis of AIDS and develops in nearly 15% of all cases. Current chemotherapy regimens are associated with substantial toxicity, particularly bone marrow suppression, which limit their long-term use. In an attempt to reduce treatment-related toxicity and enhance uptake of the drug in tumor cells, free and liposome-encapsulated doxorubicin was tested in vitro. The liposomes were prepared with cardiolipin, phosphatidylcholine, and cholesterol. Kaposi's sarcoma (KS)-derived spindle cells were exposed to free doxorubicin (DOX) and liposome-encapsulated doxorubicin (LED) for various time intervals and analyzed for cellular cytotoxicity, thymidine incorporation, and cellular drug uptake. Cytotoxicity studies of KS cells with free DOX and LED showed an IC50 of 288 and 7.5 ng/ml, respectively, hence demonstrating a 38-fold higher cytotoxicity by LED. Thymidine incorporation studies in KS cells demonstrated over one log higher toxicity to LED compared to free DOX. Cellular drug uptake studies showed that free DOX concentration peaked in 1 hr in KS cells whereas LED continued to accumulate up to 4 hr. At 4 hr, anthracycline uptake through LED was fivefold higher than the uptake of free drug. Similarly LED uptake in the cells evaluated by direct fluorescent microscopy was much more intense and more frequent than the uptake of free drug. Thus AIDS-KS cells appear to be exquisitely sensitive to LED, which may provide a higher therapeutic to toxicity index in clinical use.

Inhibition of AIDS-associated Kaposi's sarcoma cell growth by DAB389-interleukin 6.

Acquired immune deficiency syndrome-associated Kaposi's sarcoma (AIDS-KS)-derived spindle cells produce and use interleukin 6 (IL-6) among several other cytokines as a growth factor. In this study we show that AIDS-KS cells express approximately 1100 high-affinity IL-6 receptors (IL-6R) per cell with a dissociation constant (Kd) of 110 pM. Furthermore, AIDS-KS cells express the IL-6R alpha subunit, detected as a single 5.0-kb messenger ribonucleic acid species, and the high-affinity converting, signal-transducing IL-6R beta subunit designated as gp130. Similarly, tumor tissue obtained from patients with KS and AIDS expresses IL-6R messenger ribonucleic acid. We have exploited the chimeric fusion toxin DAB389-IL-6, which exerts cellular toxicity only to the cells expressing IL-6R. This chimeric protein was engineered by fusion of a truncated diphtheria toxin structural gene, in which the region encoding the native receptor-binding domain was removed and replaced with the gene encoding IL-6. DAB389-IL-6 inhibited protein synthesis in AIDS-KS-derived spindle cells at very low concentrations (IC50 of 3.4 x 10(-11) M). Similarly, inhibition of cell viability by DAB389-IL-6 was observed at equivalent dose levels (IC50 of 5 x 10(-11)). These effects on protein synthesis and cell viability can be abrogated by recombinant human IL-6, indicating receptor specificity. Thus, DAB389-IL-6 is a potential agent for the treatment of AIDS-associated KS.