Comparison of Pyrrolobenzodiazepine Dimer Bis-imine versus Mono-imine: DNA Interstrand Cross-linking, Cytotoxicity, Antibody-Drug Conjugate Efficacy and Toxicity.

Antibody-drug conjugates (ADC) delivering pyrrolobenzodiazepine (PBD) DNA cross-linkers are currently being evaluated in clinical trials, with encouraging results in Hodgkin and non-Hodgkin lymphomas. The first example of an ADC delivering a PBD DNA cross-linker (loncastuximab tesirine) has been recently approved by the FDA for the treatment of relapsed and refractory diffuse large B-cell lymphoma. There has also been considerable interest in mono-alkylating PBD analogs. We conducted a head-to-head comparison of a conventional PBD bis-imine and a novel PBD mono-imine. Key Mitsunobu chemistry allowed clean and convenient access to the mono-imine class. Extensive DNA-binding studies revealed that the mono-imine mediated a type of DNA interaction that is described as "pseudo cross-linking," as well as alkylation. The PBD mono-imine ADC demonstrated robust antitumor activity in mice bearing human tumor xenografts at doses 3-fold higher than those that were efficacious for the PBD bis-imine ADC. A single-dose toxicology study in rats demonstrated that the MTD of the PBD mono-alkylator ADC was approximately 3-fold higher than that of the ADC bearing a bis-imine payload, suggesting a comparable therapeutic index for this molecule. However, although both ADCs caused myelosuppression, renal toxicity was observed only for the bis-imine, indicating possible differences in toxicologic profiles that could influence tolerability and therapeutic index. These data show that mono-amine PBDs have physicochemical and pharmacotoxicologic properties distinct from their cross-linking analogs and support their potential utility as a novel class of ADC payload.

Efficacy of factory-treated and dip-it-yourself long lasting insecticide-treated bednets against cutaneous leishmaniasis vectors in the sub-Andean region of Colombia: results after two years of use.

BACKGROUND: Long lasting insecticide-treated nets (LLINs) may be effective for vector control of cutaneous leishmaniasis (CL). Their efficacy, however, has not been sufficiently evaluated. OBJECTIVE: To evaluate the large-scale efficacy of LLINs on Lutzomyia longiflocosa entomological parameters up to two years post-intervention in the sub-Andean region of Colombia. METHODS: A matched-triplet cluster-randomised study of 21 rural settlements, matched by pre-intervention L. longiflocosa indoor density was used to compare three interventions: dip it yourself (DIY) lambda-cyhalothrin LLIN, deltamethrin LLIN, and untreated nets (control). Sand fly indoor density, feeding success, and parity were recorded using CDC light trap collections at 1, 6, 12, and 24 months post-intervention. FINDINGS: Both LLINs reduced significantly (74-76%) the indoor density and the proportion of fully engorged sand flies up to two years post-intervention without differences between them. Residual lethal effects of both LLINs and the use of all nets remained high throughout the two-year evaluation period. CONCLUSIONS: Both LLINs demonstrated high efficacy against L. longiflocosa indoors. Therefore, the deployment of these LLINs could have a significant impact on the reduction of CL transmission in the sub-Andean region. The DIY lambda-cyhalothrin kit may be used to convert untreated nets to LLINs increasing coverage.

TR1801-ADC: a highly potent cMet antibody-drug conjugate with high activity in patient-derived xenograft models of solid tumors.

cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and, more recently, antibody-drug conjugates (ADCs). However, the clinical benefit from cMet-targeted therapy has been limited. We developed a novel cMet-targeted 'third-generation' ADC, TR1801-ADC, that was optimized at different levels including specificity, stability, toxin-linker, conjugation site, and in vivo efficacy. Our nonagonistic cMet antibody was site-specifically conjugated to the pyrrolobenzodiazepine (PBD) toxin-linker tesirine and has picomolar activity in cancer cell lines derived from different solid tumors including lung, colorectal, and gastric cancers. The potency of our cMet ADC is independent of MET gene copy number, and its antitumor activity was high not only in high cMet-expressing cell lines but also in medium-to-low cMet cell lines (40 000-90 000 cMet/cell) in which a cMet ADC with tubulin inhibitor payload was considerably less potent. In vivo xenografts with low-medium cMet expression were also very responsive to TR1801-ADC at a single dose, while a cMet ADC using a tubulin inhibitor showed a substantially reduced efficacy. Furthermore, TR1801-ADC had excellent efficacy with significant antitumor activity in 90% of tested patient-derived xenograft models of gastric, colorectal, and head and neck cancers: 7 of 10 gastric models, 4 of 10 colorectal cancer models, and 3 of 10 head and neck cancer models showed complete tumor regression after a single-dose administration. Altogether, TR1801-ADC is a new generation cMet ADC with best-in-class preclinical efficacy and good tolerability in rats.

Efficacy of factory-treated and dip-it-yourself long lasting insecticide-treated bednets against cutaneous leishmaniasis vectors in the sub-Andean region of Colombia: results after two years of use

BACKGROUND Long lasting insecticide-treated nets (LLINs) may be effective for vector control of cutaneous leishmaniasis (CL). Their efficacy, however, has not been sufficiently evaluated. OBJECTIVE To evaluate the large-scale efficacy of LLINs on Lutzomyia longiflocosa entomological parameters up to two years post-intervention in the sub-Andean region of Colombia. METHODS A matched-triplet cluster-randomised study of 21 rural settlements, matched by pre-intervention L. longiflocosa indoor density was used to compare three interventions: dip it yourself (DIY) lambda-cyhalothrin LLIN, deltamethrin LLIN, and untreated nets (control). Sand fly indoor density, feeding success, and parity were recorded using CDC light trap collections at 1, 6, 12, and 24 months post-intervention. FINDINGS Both LLINs reduced significantly (74-76%) the indoor density and the proportion of fully engorged sand flies up to two years post-intervention without differences between them. Residual lethal effects of both LLINs and the use of all nets remained high throughout the two-year evaluation period. CONCLUSIONS Both LLINs demonstrated high efficacy against L. longiflocosa indoors. Therefore, the deployment of these LLINs could have a significant impact on the reduction of CL transmission in the sub-Andean region. The DIY lambda-cyhalothrin kit may be used to convert untreated nets to LLINs increasing coverage.

Experimental hut to study the indoor behaviour and effects of insecticide-treated bednets on phlebotomine sand flies (Diptera: Psychodidae).

INTRODUCTION: Behavioural effects of insecticides on endophagic phlebotomine sand fly vectors of Leishmania are poorly understood mainly because of the lack of an experimental hut (EH) in which to study them. OBJECTIVE: To build an EH to evaluate the effects of long-lasting insecticide-treated nets (LLINs) on Lutzomyia longiflocosa. METHODS: The study had two phases: (1) Laboratory experiments using tunnel tests to select the traps for the EH; and (2) EH construction and evaluation of the effects of deltamethrin and lambda-cyhalothrin LLINs on L. longiflocosa females inside the EH. FINDINGS: Phase 1: The horizontal-slit trap was the best trap. This trap collected the highest percentage of sand flies, and prevented them from escaping. Therefore, this trap was used in the EH. Phase 2: The main effects of LLINs on L. longiflocosa in the EH were: landing inhibition, inhibition from entering the bednet, induced exophily, and high mortality (total and inside exit traps). CONCLUSIONS: The EH was effective for evaluating the effects of LLINs on endophagic sand flies. Although both types of LLINs showed high efficacy, the lambda-cyhalothrin-treated LLIN performed better. This is the first report of induced exophily in sand flies.

Experimental hut to study the indoor behaviour and effects of insecticide-treated bednets on phlebotomine sand flies (Diptera: Psychodidae)

BACKGROUND Behavioural effects of insecticides on endophagic phlebotomine sand fly vectors of Leishmania are poorly understood mainly because of the lack of an experimental hut (EH) in which to study them. OBJECTIVE To build an EH to evaluate the effects of long-lasting insecticide-treated nets (LLINs) on Lutzomyia longiflocosa. METHODS The study had two phases: (1) Laboratory experiments using tunnel tests to select the traps for the EH; and (2) EH construction and evaluation of the effects of deltamethrin and lambda-cyhalothrin LLINs on L. longiflocosa females inside the EH. FINDINGS Phase 1: The horizontal-slit trap was the best trap. This trap collected the highest percentage of sand flies, and prevented them from escaping. Therefore, this trap was used in the EH. Phase 2: The main effects of LLINs on L. longiflocosa in the EH were: landing inhibition, inhibition from entering the bednet, induced exophily, and high mortality (total and inside exit traps). CONCLUSIONS The EH was effective for evaluating the effects of LLINs on endophagic sand flies. Although both types of LLINs showed high efficacy, the lambda-cyhalothrin-treated LLIN performed better. This is the first report of induced exophily in sand flies.

Entering and exiting behaviour of the phlebotomine sand fly Lutzomyia longiflocosa (Diptera: Psychodidae) in rural houses of the sub-Andean region of Colombia

The present study identified the entering and exiting sites for Lutzomyia longiflocosa in rural houses of the sub-Andean region in Colombia. Entering sites were identified with sticky traps set up outside the bedrooms, around the eave openings, and with cage traps enclosing the slits in the doors and windows inside the bedrooms. Exiting sites were identified by releasing groups of females indoors. These females were blood fed and marked with fluorescent powders. Females were recaptured with the trap placement described above but set up on the opposite sides of the openings. In the entering experiment, a significantly higher number of females were captured in the sticky traps at the zone nearest the eave openings (n = 142) than those captured in the other zones of the trap (n = 52); similarly, a higher number of females were captured on the front side of the house (n = 105) than at the rear side (n = 37). Only two females were collected in the cage trap. In the exiting experiment, at the ceiling, the highest percentage (86.2%) of females was recaptured with sticky traps nearest the eave openings and on the front side of the house (70.0%). Seven females were collected in the cage trap. Lu. longiflocosa entered and exited houses primarily through the eave openings in a non-random pattern in relation to the sides of the house.

Entering and exiting behaviour of the phlebotomine sand fly Lutzomyia longiflocosa (Diptera: Psychodidae) in rural houses of the sub-Andean region of Colombia.

The present study identified the entering and exiting sites for Lutzomyia longiflocosa in rural houses of the sub-Andean region in Colombia. Entering sites were identified with sticky traps set up outside the bedrooms, around the eave openings, and with cage traps enclosing the slits in the doors and windows inside the bedrooms. Exiting sites were identified by releasing groups of females indoors. These females were blood fed and marked with fluorescent powders. Females were recaptured with the trap placement described above but set up on the opposite sides of the openings. In the entering experiment, a significantly higher number of females were captured in the sticky traps at the zone nearest the eave openings (n = 142) than those captured in the other zones of the trap (n = 52); similarly, a higher number of females were captured on the front side of the house (n = 105) than at the rear side (n = 37). Only two females were collected in the cage trap. In the exiting experiment, at the ceiling, the highest percentage (86.2%) of females was recaptured with sticky traps nearest the eave openings and on the front side of the house (70.0%). Seven females were collected in the cage trap. Lu. longiflocosa entered and exited houses primarily through the eave openings in a non-random pattern in relation to the sides of the house.

The Pro-apoptotic STK38 Kinase Is a New Beclin1 Partner Positively Regulating Autophagy.

Autophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated. Here, we describe the STK38 protein kinase (also termed NDR1) as a conserved regulator of autophagy. Using STK38 as bait in yeast-two-hybrid screens, we discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological, and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 activity, as judged by PI3P formation. Furthermore, we observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, we found that STK38 is stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, our data establish STK38 as a conserved regulator of autophagy in human cells and flies. We also provide evidence demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions.

Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation.

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease.

KIR-HLA clase I y tuberculosis pulmonar en población amerindia del Chaco, Argentina / KIR-HLA class I and pulmonary tuberculosis in the Amerindian population in Chaco, Argentina

INTRODUCCIÓN: La susceptibilidad a la tuberculosis pulmonar (TB) es multifactorial, por lo que factores genéticos como las moléculas del complejo mayor de histocompatibilidad (CMH) y los receptores tipo inmunoglobulinas presentes en células NK (KIR) podrían predisponer al desarrollo de la misma. OBJETIVO: Evaluar si algún alelo de HLA clasei en combinación con determinados KIR podría estar relacionado con el desarrollo de TB en la comunidad amerindia Wichi en el noreste argentino. MÉTODOS: En un estudio de cohorte se incluyeron 18 familias, 35 individuos afectados con TB, 84 convivientes familiares y 63 controles sanos del mismo grupo étnico. Los loci A y B de HLA clase I se tipificaron mediante amplificación genérica seguida de hibridación reversa (Dynal), el locus C por PCR-SSOP. Los receptores KIR fueron amplificados con primers de secuencia específica SSP-PCR. RESULTADOS: Se observó una asociación altamente significativa con el alelo B*35:19/47 en TB vs. contactos familiares [Pc = 0,0051] y vs. controles [Pc = 0,0033] y con el alelo HLA-C*03 en TB vs. contactos [Pc = 0,014] y vs. controles [Pc = 0,0033]. Cuando se analizaron los receptores KIR, se observó aumento de la frecuencia KIR2DL3/KIR2DL3 en combinación con el grupo C1 de HLA-C (p = 0,018). C*03 pertenece al grupo C1, por lo que podemos pensar que esta combinación ejerza una fuerte acción inhibitoria sobre la célula infectada con Mycobacterium. CONCLUSIÓN: HLA-B35:19/47-HLA-C*3 podrían ser un factor de susceptibilidad a TB y la combinación KIR2DL3-HLA-C1, por su efecto inhibitorio sobre las células NK, podría contribuir al curso clínico de la infección por M. tuberculosis

INTRODUCTION: The susceptibility to pulmonary tuberculosis (TB) is multifactorial, thus genetic factors such as HLA and immunoglobulins-like killer receptors (KIR) could be predisposed to the development of the disease. Aim To evaluate whether any HLA class I were typed by generic PCR followed by reverse hybridization (Dynal), locus C by PCR-SSOP. KIR receptors were studied using sequence specific PCR. RESULTS: There was a highly significant association with allele B*35:19/47 in TB vs. household contacts [Pc = 0.0051] and vs. controls [Pc = 0.0033], and with allele HLA-C*03 in TB vs. household contacts [Pc = 0.014] and vs. controls [Pc=0.0033]. KIR receptors had shown increased KIR2DL3/KIR2DL3 frequency in combination with the C1 group of HLA-C (P = .018). HLA-C*03 belongs to C1 group, and this combination could have a strong inhibitory action on the infected cell. CONCLUSION: HLA-B35:19/47-C*03 haplotype could be a susceptibility factor to TB and KIR2DL3-HLA-C1 combination have an inhibitory capacity on NK cells, and might contribute to the course of the infection by Mycobacterium tuberculosis

[KIR-HLA class i and pulmonary tuberculosis in the Amerindian population in Chaco, Argentina]. / KIR-HLA clase i y tuberculosis pulmonar en población amerindia del Chaco, Argentina.

INTRODUCTION: The susceptibility to pulmonary tuberculosis (TB) is multifactorial, thus genetic factors such as HLA and immunoglobulins-like killer receptors (KIR) could be predisposed to the development of the disease. Aim To evaluate whether any HLA classi allele and its combination with KIR could be related to the development of TB in the Wichi Amerindian community in north-eastern Argentina. METHODS: A cohort study was conducted that included 18 families, 35 individuals affected with TB, 84 cohabiting families, and 63 controls of the same ethnic group. A and B loci of HLA classi were typed by generic PCR followed by reverse hybridization (Dynal), locus C by PCR-SSOP. KIR receptors were studied using sequence specific PCR. RESULTS: There was a highly significant association with allele B*35:19/47 in TB vs. household contacts [Pc=0.0051] and vs. controls [Pc=0.0033], and with allele HLA-C*03 in TB vs. household contacts [Pc=0.014] and vs. controls [Pc=0.0033]. KIR receptors had shown increased KIR2DL3/KIR2DL3 frequency in combination with the C1 group of HLA-C (P=.018). HLA-C*03 belongs to C1 group, and this combination could have a strong inhibitory action on the infected cell. CONCLUSION: HLA-B35:19/47-C*03 haplotype could be a susceptibility factor to TB and KIR2DL3-HLA-C1 combination have an inhibitory capacity on NK cells, and might contribute to the course of the infection by Mycobacterium tuberculosis.

[Field efficacy of repellent formulation containing para-menthane-3,8-diol and lemongrass against Culicoides pachymerus (Diptera: Ceratopogonidae) in Colombia]. / Eficacia en campo de un repelente a base de para-mentano-3,8-diol y aceite de limonaria contra Culicoides pachymerus (Diptera: Ceratopogonidae) en Colombia.

INTRODUCTION: Culicoides pachymerus is a major pest species for the inhabitants of the western Boyacá province of Colombia. OBJECTIVE: The effect of a repellent lotion based on p-menthane-3,8-diol (16%) and lemongrass oil (2%) was evaluated against the bites of C. pachymerus. MATERIALS AND METHODS: The repellent lotion was compared simultaneously with a control (no treatment) by human landing catches of C. pachymerus on the forearms of paired volunteers situated near human dwellings. Protection percentage and protection time for 3 to 6 h after repellent application was calculated. The test was repeated ten times. RESULTS: Only two females of C. pachymerus were collected on arms with the repellent treatment. In contrast, the mean biting rate in the untreated control was 47.7 midges/person/10 min. Mean protection percentage of the repellent was 100% up to 4 h and 99.5% up to 5 h. Protection time was 332.2 and 338.2 min in the two replicates where bites of C. pachymerus were confirmed. In the remaining eight replicates protection time exceeded the test duration. CONCLUSION: The repellent showed high efficacy against C. pachymerus, up to 5 h post-application.

pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking.

BACKGROUND: Huntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntington's disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease. RESULTS: We describe a flexible synthetic vector encoding full-length htt called pARIS-htt (Adaptable, RNAi Insensitive &Synthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells. CONCLUSION: We report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its structure/function. This may help provide relevant information about the cellular dysfunctions operating during the disease. As proof of principle, we show that either polyQ expansion or deletion of key interacting domains within full-length htt protein impairs its function in transport indicating that HD mutation induces defects on intrinsic properties of the protein and further demonstrating the importance of studying htt in its full-length context.

Breeding sites of Culicoides pachymerus Lutz in the Magdalena River basin, Colombia.

The breeding sites of Culicoides pachymerus are described for the first time in western Boyacá Province, Colombia, where this species is a public health problem. In addition to being a nuisance due to its enormous density and its high biting rates, C. pachymerus cause dermatological problems in the human population. Analysis of microhabitats by the sugar flotation technique and the use of emergence traps allowed us to recover 155 larvae of Culicoides spp and 65 adults of C. pachymerus from peridomiciliary muddy substrates formed by springs of water and constant rainwater accumulation. These important findings could aid in the design of integrated control measures against this pest.

Breeding sites of Culicoides pachymerus Lutz in the Magdalena River basin, Colombia

The breeding sites of Culicoides pachymerus are described for the first time in western Boyacá Province, Colombia, where this species is a public health problem. In addition to being a nuisance due to its enormous density and its high biting rates, C. pachymerus cause dermatological problems in the human population. Analysis of microhabitats by the sugar flotation technique and the use of emergence traps allowed us to recover 155 larvae of Culicoides spp and 65 adults of C. pachymerus from peridomiciliary muddy substrates formed by springs of water and constant rainwater accumulation. These important findings could aid in the design of integrated control meas-ures against this pest.