SARS-CoV-2 RNA screening in routine pathology specimens

Virus detection methods are important to cope with the SARS-CoV-2 pandemics. Apart from the lung, SARS-CoV-2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom-indicated swab testing. Here, we tested and validated several approaches and selected the most reliable RT-PCR protocol for the detection of SARS-CoV-2 RNA in patients’ routine diagnostic formalin-fixed and paraffin-embedded (FFPE) specimens available in pathology, to assess (i) organ tropism in samples from COVID-19-positive patients, (ii) unrecognized cases in selected tissues from negative or not-tested patients during a pandemic peak, and (iii) retrospectively, pre-pandemic lung samples. We identified SARS-CoV-2 RNA in seven samples from confirmed COVID-19 patients, in two gastric biopsies, one small bowel and one colon resection, one lung biopsy, one pleural resection and one pleural effusion specimen, while all other specimens were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID-19 case in tonsillectomy samples. All pre-pandemic lung samples were negative. In conclusion, SARS-CoV-2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID-19, allow retrospective studies, and advance our understanding of SARS-CoV-2 organ tropism and effects.     

Artificial endosperm of Cleopatra tangerine zygotic embryos: a model for somatic embryo encapsulation

Synthetic seed technology may be of value in breeding programs and allow the propagation of many elite genotype-derived plants in a short time. In this work, a range of artificial endosperm treatments of Cleopatra tangerine zygotic embryos were evaluated for suitability for encapsulation of somatic embryos. Different complexing ions in the form of alginate capsules, zeolite as an ion exchanger and the relationship between capsule-nutrient gel on germination of zygotic embryos, were evaluated. Artificial endosperm assays showed that abscisic acid (1 μM) and mannitol (0.25 M) delayed germination and conversion of zygotic embryos, whereas amino acid supplements (proline, glutamic acid and arginine) accelerated the conversion process. An artificial endosperm was used to encapsulate somatic and zygotic embryos. After encapsulation, zygotic embryos germinated after four days of culture while somatic embryos germinated asynchronously after 20 days. Somatic embryo-derived plantlets showed greater vigour than zygotic embryo-derived plantlets. Results showed that this artificial endosperm is adequate for Cleopatra tangerine somatic embryo germination and conversion into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

DCDC2 Mutations Cause a Renal-Hepatic Ciliopathy by Disrupting Wnt Signaling

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits β-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC.     

Field performance of artificial seed-derived sugarcane plants

This study shows the behaviour of sugarcane plants cv. CP-5243 derived from artificial seed compared with traditional and isolated bud methods. Artificial seed-acclimatised plants were planted in field conditions simultaneously with two-control treatments previously germinated: macropropagated plants derived from stems of three buds and axillary buds isolated from field-grown plants. Plants from artificial seed were taller and had a smaller diameter at 8 months, but these differences disappeared at 12 months. With respect to sugar analysis and yield, no differences in all parameters evaluated were found between artificial seed-derived plants and plants derived from the two other methods.     

Storage proteins in sugarcane: An interesting exception in monocots

Storage proteins were extracted from sugarcane seeds, globular embryos (formed on embryogenic calluses and collected in early developmental stages) and embryogenic cells. In all cases, the major percentage of storage proteins were albumins and globulins and the lower percentage were prolamins and glutelins. Sugarcane is an interesting exception in monocots which usually have high levels of prolamins and glutelins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biochemical characterization of embryogenic and non-embryogenic calluses of sugarcane

Summary With the aim to determine a possible relationship between somatic embryogenesis and some metabolic contents in embryogenic and non-embryogenic calluses of sugarcane (Saccharum sp. var CP-5243), the present study was carried out. Embryogenic callus has more soluble proteins, free proline, proteolytic activity, soluble sugars, and invertase, and lower putreseine/(spermidine + spermine) than non-embryogenic tissue. Non-embryogenic callus has a higher peroxidase and gallic acid level, lower dry matter/fresh matter ratio, and more gross fat compared with embryogenic callus.     

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei

The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre‐cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post‐mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post‐mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol‐anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans‐face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi.      

Effect of abscisic acid and jasmonic acid on partial desiccation of encapsulated somatic embryos of sugarcane

Embryogenic calli of sugarcane (Saccharum sp. hybrid, clone CP52-43), with somatic embryos in the late scutelar stage, were subjected to different treatments for increasing embryo tolerance to desiccation. The medium was supplemented with abscisic acid (ABA) (3.8 μM), jasmonic acid (JA) (4.7 μM) or a combination of them. A control treatment without growth regulators was also included. The embryos were encapsulated in alginate beads and dehydrated or not in sucrose (0.5 M). Thereafter, they were further dehydrated in chambers containing silicagel until the beads reached either 60% or 30% of water content (WC). Survival of encapsulated-dehydrated embryos was achieved only in the control and ABA treatment. ABA induced an increase in protein, polyamines, free proline levels and starch levels as a response to desiccation tolerance. JA treatment showed the lowest protein and polyamines levels and increased the starch content almost two-fold compared to the ABA treatment. The JA treatment induced high levels of 4-methylcatechol and the lowest levels of gallic acid. However, the ABA treatment increased gallic acid and p-coumaric acid content in the induction medium. Some differences were found in growth regulator free-medium in relation to the induction medium. JA is not effective in these desiccation processes. The mechanisms by which these two plant growth regulators act on the induction of tolerance to stress are presumably different. This revised version was published online in June 2006 with corrections to the Cover Date.