3'dNTP Binding Is Modulated during Primer Synthesis and Translesion by Human PrimPol.

PrimPol is a DNA primase/polymerase from the Archaeo-Eukaryotic Primase (AEP) superfamily that enables the progression of stalled replication forks by synthesizing DNA primers ahead of blocking lesions or abnormal structures in the ssDNA template. PrimPol's active site is formed by three AEP-conserved motifs: A, B and C. Motifs A and C of human PrimPol (HsPrimPol) harbor the catalytic residues (Asp114, Glu116, Asp280) acting as metal ligands, whereas motif B includes highly conserved residues (Lys165, Ser167 and His169), which are postulated to stabilize 3' incoming deoxynucleotides (dNTPs). Additionally, other putative nucleotide ligands are situated close to motif C: Lys297, almost invariant in the whole AEP superfamily, and Lys300, specifically conserved in eukaryotic PrimPols. Here, we demonstrate that His169 is absolutely essential for 3'dNTP binding and, hence, for both primase and polymerase activities of HsPrimPol, whereas Ser167 and Lys297 are crucial for the dimer synthesis initiation step during priming, but dispensable for subsequent dNTP incorporation on growing primers. Conversely, the elimination of Lys165 does not affect the overall primase function; however, it is required for damage avoidance via primer-template realignments. Finally, Lys300 is identified as an extra anchor residue to stabilize the 3' incoming dNTP. Collectively, these results demonstrate that individual ligands modulate the stabilization of 3' incoming dNTPs to optimize DNA primer synthesis efficiency during initiation and primer maturation.

First report of Neoscytalidium dimidiatum causing stem canker on yellow dragon fruit (Hylocereus megalantus) in Ecuador.

Dragon fruit cultivation is an emerging industry in Ecuador. In August of 2020, yellow dragon fruit plants (Hylocereus megalantus) showing brown hardened scabs on cladodes and fruits were observed in a field in Guayas, a coastal province of Ecuador (Fig. 1A). Symptoms were observed in ~ 40% of the assessed plants (n=100) with damage varying from mild (necrotic spots) to severe (canker). Ten cladode sections of ~ 10 cm2 with signs of canker were collected from five affected plants (two from each plant). Symptomatic cladode sections were sliced, surface-sterilized with a solution of 1% sodium hypochlorite, rinsed with sterile water, transferred into potato dextrose agar (PDA) media, and incubated at 28°C for five days in the dark. Three fungal isolates recovered from the cladodes produced colonies with dense dark aerial mycelia that matched the morphological description for Neoscytalidium dimidiatum (Crous et al 2006) (Fig 1B, 1C). Arthric chains of cylindrical conidia were observed under the microscope and presented zero or one septum with sizes between 10.9 ± 0.27 x 4.97 ± 0.36 µm (n=50). Pycnidia produced ellipsoid-shaped conidia, and sizes ranged from 4.5 ± 0.3 x 11.02 ± 0.5 µm (n=50). Cultured isolates were subjected to DNA extractions using the fungal DNA mini kit (Omega, Bio-Tek, Inc) for molecular identification by amplifying the 5.8S rDNA and adjacent internal transcriber spacer (ITS) 1 and 2 regions using primers (ITS1 / ITS4) as described (White et al., 1990). In addition, the ß-tubulin and elongation factor 1-α targets were amplified by primers Bt2a / Bt2b (Glass and Donaldson, 1995) and EF1-728 F / EF1-986R (Carbone and Kohn, 1999), respectively, following the recommended PCR conditions. Amplified products were cloned using a pGEMT-easy kit (Promega, USA) and sequenced. Sequence comparisons for each target revealed that the three isolates were 100% identical to each other. Sequences obtained from a single plant were submitted to NCBI Genbank and assigned acc. Numbers OP377444, OP381216, and OP381217, for the ITS, ß-tubulin, and elongation factor, respectively. BLAST analyses of the three amplified targets confirmed homology to counterparts from N. dimitiatum, with 99-100 % identities to isolates from China (JX524168), United Arab Emirates (MN447201), and Israel (KF020895). To fulfill Koch´s postulates, thirteen 4-month-old healthy plants were inoculated either with the pathogen (n=10) or mock-inoculated (n=3). In addition, two detached yellow dragon fruits and a mock were inoculated following the same protocol as stems. Inoculation was performed by making small wounds on cladodes or fruits using a sterile needle and placing a 2-mm agar plug containing mycelia from a 7-day-old colony. For mock inoculations, 2-mm plugs with clean PDA media were used. The inoculated area was wrapped with black plastic film for seven days at 27 to 32°C. At ten days post-inoculation, brown scab lesions with an orange halo were observed in the inoculated plants but not in the mock-inoculated plants and fruit (Fig. 1D, 1E). N. dimidiatum was re-isolated from experimentally-induced canker lesions, and morphologically identified. Stem canker caused by N. dimidiatum is a severe disease affecting dragon fruit production worldwide (Chuang et al., 2021). In Ecuador, this pathogen was found affecting yellow dragon fruit (H. megalantus) under natural conditions; however, under experimental conditions, red dragon fruit (H. undatus), which is widely produced in several coastal provinces of the country, was also found to be susceptible. Further studies are needed to investigate epidemiological aspects of this important pathogen, which threatens dragon fruit production in Ecuador.

Membrane Vesicles for Nanoencapsulated Sulforaphane Increased Their Anti-Inflammatory Role on an In Vitro Human Macrophage Model.

At present, there is a growing interest in finding new non-toxic anti-inflammatory drugs to treat inflammation, which is a key pathology in the development of several diseases with considerable mortality. Sulforaphane (SFN), a bioactive compound derived from Brassica plants, was shown to be promising due to its anti-inflammatory properties and great potential, though its actual clinical use is limited due to its poor stability and bioavailability. In this sense, the use of nanocarriers could solve stability-related problems. In the current study, sulforaphane loaded into membrane vesicles derived from broccoli plants was studied to determine the anti-inflammatory potential in a human-macrophage-like in vitro cell model under both normal and inflammatory conditions. On the one hand, the release of SFN from membrane vesicles was modeled in vitro, and two release phases were stabilized, one faster and the other slower due to the interaction between SFN and membrane proteins, such as aquaporins. Furthermore, the anti-inflammatory action of sulforaphane-loaded membrane vesicles was demonstrated, as a decrease in interleukins crucial for the development of inflammation, such as TNF-α, IL-1ß and IL-6, was observed. Furthermore, these results also showed that membrane vesicles by themselves had anti-inflammatory properties, opening the possibility of new lines of research to study these vesicles, not only as carriers but also as active compounds.

A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol.

Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.

Trauma dentario en una adolescente / Dental trauma in an adolescent

Se describe el caso clínico de una paciente de 15 años de edad, quien producto de un trauma, sufrió luxación intrusiva del 12 y lateral del 11 con pérdida de la vitalidad de ambos dientes. Recibió tratamiento inmediato en cuerpo de guardia con lenta evolución y debido a otras manifestaciones clínicas acudió a la consulta de Estomatología General Integral de la Clínica Estomatológica Provincial Docente Mártires del Moncada de Santiago de Cuba. Se le realizó tratamiento endodóntico en ambos dientes y fue remitida a la consulta de Ortodoncia donde se le colocó aparatología fija hasta lograr un resalte y sobrepase funcional.

The case report of a 15 year-old patient is described who as a result of a trauma, suffered intrusive luxation of the 12 and lateral of the 11 with loss of vitality of both teeth. She received immediate treatment in the emergency room with slow course and due to other clinical manifestations she visited the Comprehensive General Stomatology service of Mártires del Moncada Teaching Provincial Stomatological Clinic in Santiago de Cuba . Endodontic treatment was carried out in both teeth and it was referred to the Orthodontics service where fixed aparatology was placed up to achieving a functional resalte y sobrepase.

Exogenous melatonin supplementation prevents oxidative stress-evoked DNA damage in human spermatozoa.

Reactive oxygen species (ROS) are essential for sperm physiological functions such as capacitation, hyperactivation, and acrosome reaction, on the one hand, and for stimulating the apoptotic processes involved in the regulation of spermatogenesis, on the other hand. However, the imbalance between production and removal of ROS leads to oxidative stress, which is referred to as one of the main factors involved in male infertility. The pineal hormone melatonin, given its low toxicity and well-known antioxidant capacity, could be an excellent candidate to improve sperm quality. For this reason, the objective of the present work was to analyze whether long-term supplementation with melatonin to infertile men affects human sperm quality and the quality of the embryos retrieved from their couples. Our findings showed that the daily supplementation of 6 mg melatonin, as early as after 45 days of treatment, produced an increase in melatonin endogenous levels, indirectly measured as urinary 6-sulfatoxymelatonin (aMT6-s), an enhancement of both urinary and seminal total antioxidant capacity, and a consequent reduction in oxidative damage caused in sperm DNA. Moreover, couples whose men were given melatonin showed a statistically significant increase in the percentage of grade A (embryo with blastomeres of equal size; no cytoplasmic fragmentation), B (embryo with blastomeres of equal size; minor cytoplasmic fragmentation), and C (embryo with blastomeres of distinctly unequal size; significant cytoplasmic fragmentation) embryos at the expense of grade D (embryo with blastomeres of equal or unequal size; severe or complete fragmentation.) embryos which were clearly reduced. In summary, melatonin supplementation improves human sperm quality, which is essential to achieve successful natural and/or assisted reproduction outcome.

Unsuspected urinoma of an obstructed kidney detected in a bone scan in a patient with lung cancer.

A 60-year-old woman with an adenocarcinoma of the lung was referred to our department for a bone scan to assess the extension of the metastatic bone disease. The images showed several foci of radiotracer uptake in the bone consistent with malignant involvement. Besides, a considerable accumulation of tracer was shown in the right perinephric region as well as in the renal pelvis. SPECT/CT images demonstrated a superior perirenal urine leak due to rupture of the collecting system. The leakage was treated by urinary catheter decompression with complete recovery after 4 weeks.

Mutual regulation of hypoxic and retinoic acid related signalling in tubular proximal cells.

Hypoxia-inducible factor-1α (HIF-1α) and all-trans retinoic acid (ATRA) afford protection in several experimental models of kidney disease. HIF-1α protein is degraded under normoxia but stabilized by hypoxia, which activates its transcription factor function. ATRA activates another set of transcription factors, the retinoic acid receptors (RAR) α, ß and γ, which mediate its effects on target genes. ATRA also up-regulates the expression of RAR α, ß and γ at the transcriptional level. Here we demonstrate the presence of mutual regulation of hypoxic and retinoic acid related signalling in tubular proximal cells. In human proximal tubular HK-2 cells we have found that: (i) ATRA treatment induces HIF-1α under normoxic conditions and also synergizes with hypoxia leading to the over-expression of HIF-1α and vascular endothelial growth factor-A, a HIF-1α-regulated renal protector. ATRA-induced HIF-1α expression involved stabilization of HIF-1α mRNA but not of HIF-1α protein. (ii) Expression of HIF-1α is an absolute requirement for the transcriptional up-regulation of RARß by ATRA. Transfection with HIF-1α siRNA abolished the induction by ATRA of the expression of both RARß mRNA and protein while treatment with HIF-1α inhibitor YC-1 results in the abolishment of ATRA-induced activity of a retinoic acid-response element (RARE) construct from the RARß promoter. (iii) Hypoxia up-regulates RARß through HIF-1α since this effect was inhibited by HIF-1α knockdown. In contrast to ATRA-induced RARß up-regulation, induction of RARß expression by ATRA did not involve transcriptional up-regulation as hypoxia did not increase the expression of RARß mRNA or the activity of the RARE construct. These results suggest the presence of crosstalk between hypoxia/HIF-1α and ATRA/RARß that may be physiologically and pharmacologically relevant.

The enemy as ally: herbivore-induced increase in crop yield.

There is increasing global concern over the risk of food shortage and instability, and a concomitant demand for an increase in food production. However, the continuing expansion of agricultural areas threatens natural habitats as well as human and ecosystem health. One option for increasing food production is to maximize yields from existing farmland. Here we demonstrate that larval feeding by the Guatemalan potato moth (Tecia solanivora), considered one of the most economically important potato pests in Latin America, leads to a dramatic increase in potato tuber production. Field-grown potato plants (Solanum tuberosum) in the Colombian Andes attacked by low numbers of potato moth larvae produce a 2.5-fold higher marketable potato yield than undamaged plants. Greenhouse experiments demonstrate that this effect is induced by larval regurgitant, rather than by mechanical tissue damage. Our results indicate that compounds from the foregut of T. solanivora are necessary and sufficient to induce an increased yield in potato. Our study suggests that using (1) herbivore-derived chemical cues and (2) induced compensatory plant responses to herbivory can provide viable new tools to increase per area crop productivity.

Conservation and immunogenicity of the mosquito ortholog of the tick-protective antigen, subolesin.

The control of arthropod vectors of pathogens that affect human and animal health is important for the eradication of vector-borne diseases. The ortholog of the tick-protective antigen, subolesin, was identified in Aedes albopictus and found to have conserved epitopes in ticks and mosquitoes. RNA interference with the tick and mosquito double-stranded RNA in three tick species resulted in significant gene knockdown and decreased tick weight and/or survival. Feeding Anopheles atroparvus, Aedes caspius, and Culex pipiens female mosquitoes on an A. albopictus subolesin hyperimmune serum resulted in 11 +/- 5% to 29 +/- 6% survival inhibition when compared to controls fed on preimmune serum. Feeding sand flies, Phlebotomus perniciosus, on antimosquito subolesin ortholog protein antibodies inhibited female survival and the number of larvae and adults obtained after hatching by 28 +/- 22% and 16 +/- 3%, respectively, when compared to controls. Vaccination with tick and mosquito subolesin ortholog proteins significantly reduced Ixodes scapularis tick infestation and weight in a similar way. However, vaccination with the recombinant mosquito subolesin ortholog antigen did not protect against Amblyomma americanum and Rhipicephalus sanguineus tick infestations. Collectively, these preliminary results provided the first evidence that development of vaccines may be possible for control of multiple arthropod vectors using subolesin orthologs but suggested that multiple antigens may be required to produce an effective vaccine.

Evaluación de tecnologías diagnósticas: conceptos básicos en un estudio con muestreo transversal / Assessing a diagnostic test: basic concepts in a study with naturalistic sampling

Los estudios de evaluación de pruebas diagnósticas son un diseño científico que nos ayuda a determinar la validez y la reproducibilidad de los procedimientos que pueden ser usados en la práctica clínica por médicos y otros profesionales de salud. Este es un artículo de formación en epidemiología para estudiantes y profesionales de la salud y sus objetivos son: a) resumir los conceptos utilizados durante un estudio de evaluación de tecnologías diagnósticas y b) explicar cómo calcular e interpretar las medidas usadas durante la realización de un diseño con muestreo transversal, enfatizando en el significado de términos como sensibilidad, especificidad y valores predictivos de la prueba

Neonatal life-threatening arrhythmia responding to lidocaine, a probable LQTS3.

Intrauterine and neonatal manifestations of congenital long QT syndrome (LQTS) are associated with a high cardiac risk. We present a newborn, with antecedents of intrauterine premature ventricular contractions, showing in his surface electrocardiogram (ECG): a QTc of 0.69 ms, 2:1 atrioventricular block, autolimited episodes of ventricular tachycardia and Torsade de Pointes. Intravenous esmolol therapy was started, being only partially effective. Because of the ECG phenotype, a LQTS3 is suspected, and intravenous lidocaine was started, achieving sinus rhythm, a normal QTc and no new episodes of ventricular tachycardia. Lidocaine was substituted for oral mexiletine, and esmolol for propranolol. During the follow-up the patient has remained asymptomatic under therapy with propranolol and mexiletine.