The article presents the radiocarbon investigation of the baobab of Jhunsi, Allahabad and the Parijaat tree at Kintoor, two old African baobabs from northern India. Several wood samples extracted from these baobabs were analysed by using AMS radiocarbon dating. The radiocarbon date of the oldest samples were 779 ± 41 BP for the baobab of Jhunsi and 793 ± 37 BP for the baobab of Kintoor. The corresponding calibrated ages are 770 ± 25 and 775 ± 25 calendar years. These values indicate that both trees are around 800 years old and become the oldest dated African baobabs outside Africa. Adansonia digitata L.), belongs to the Bombacoideae subfamily of Malvaceae and is the best-known of the eight or nine species of the Adansonia genus [The African baobab  radiocarbon dating of tiny segments extracted from wood samples collected from inner cavities, deep incisions/entrances in the trunk, fractured stems and from the outer part/exterior of large baobabs –14.Baobabs usually start growing as single-stemmed individuals and develop into multi-stemmed trees over time, due to their ability to generate periodically new stems. By this special characteristic, old baobabs typically exhibit very complex and unexpected architectures ,13. ThusBaobab trees are considered to be a rare sight in India. However, the African baobab is relatively well distributed in states with a tropical or subtropical climate. As a result of surveys, the existence of century-old and heritage specimens has been documented in Andhra Pradesh, Karnataka, Gujarat, Madhya Pradesh, Maharashtra, Rajasthan and Uttar Pradesh. These specimens confirm an antique association of the tree with India.The introduction of the baobab from Africa to the Indian subcontinent and other regions of the Indian Ocean is a silent and historic affair. The exact route and period of introduction has not been recorded and, practically, little is known about their arrival in India. It is believed that the African baobab was brought to India especially by Arabian sailors who came to establish trade links with regions of the Indian Ocean. Portuguese, Dutch and French invasions may have contributed to the introduction of this species in India. Several studies have also speculated that baobab pods may have floated across from Africa on sea currents, reaching the Indian shores –19.Adansonia digitata. Second, there is less genetic diversity in the Indian baobab populations as compared to the African populations. This confirms the supposition that the baobabs have not been in India long enough for the populations to diversify; thus, their dispersal by ocean currents is less likely than their introduction by humans. The latter discovery indicates multiple introductions of baobabs to India and demostrates they have several biogeographical origins in Africa. Many Indian baobabs have phylogenetic relationship with baobab populations from coastal Kenya and Tanzania, some of them show closer relationship with baobabs from coastal and inland Mozambique and also from West Africa [Recent genetic research indicates multiple introduction episodes from various areas of Africa to India, supposedly dating back to prehistoric times . The gent Africa ,21.th to the 17th centuries via the Swahili-Arab trade networks. The third major interaction occured in the 17th and 19th centuries owing to the Portugese, who established their colonial bases in Mozambique and western and southern India. Finally, the fourth interaction associated with the last introduction of the African baobab to India occured over the 18th and 19th centuries, when English and Dutch colonial authorities recruited soldiers from West Africa for regiments in southern India. Thus, one can state that the geographical distribution of baobabs in India is associated with the long history of African diaspora across the Indian Ocean [By combining the genetic findings with archaeological and historical accounts of the Indian Ocean trade, Bell and Rangan identified four major periods during which Africans from different regions would have travelled to India. The earliest interactions and transportation of the African baobab to India go back more than 4,000 years ago, when cereals and legumes arrived in India from Sudan, Ethiopia and the Horn of Africa. The second interaction between Africa and India occurred from the 10an Ocean ,21.Today India hosts several large and relatively old baobabs. Recently, we investigated and dated by radiocarbon the biggest baobab outside Africa, which is located at Golconda Fort, Hyderabad, India. We found that the oldest part of this very large tree, with a girth of 25.48 m, is around 475 yr old .Here we present the radiocarbon investigation results of two sacred African baobabs from India, namely the baobab of Jhunsi, Allahabad and the Parijaat tree at Kintoor.The on-site investigation and sampling of the baobabs was authorised by the National Biodiversity Authority of India (NBA/9/2269/18/18-19). The Botanical Survey of India, Central Regional Centre of Allahabad, Uttar Pradesh, provided scientific assistance in this investigation. After each coring, the increment borer was cleaned and disinfected with methyl alcohol. The small coring holes were sealed with Steriseal (Efekto), a special polymer sealing product, for preventing any possible infection of the trees.The co-author performing the sampling in The two trees are located in northern India, in the Uttar Pradesh state. Both have a strange shape and structure, which is not typical for the African baobab.Kurma Purana and Matsya Purana as the Prayagamandala [The historic tree is situated in the village of Jhunsi (or Jhusi), a suburb of the city of Allahabad , in the Allahabad district. Radiocarbon dating relates Jhunsi to an ancient archaeological site on the left side of Ganges at Prayag, known under the name Andhernagri and Pratishthanpur. The history of the Jhunsi area begins from 7,100 BCE . Its past is documented by archaeological evidences that correspond to five cultural phases, ranging from Chalcolithic to the Early Medieval Period. Epigraphical remains discovered here during Maruya, Sunga, Kushana, Gupta and the Medieval Period testify that Jhunsi reached an advanced degree of civilization at an early date. Pratishthan (Jhunsi) is also described in amandala ,24.The baobab grows on a huge mound of mud on the left bank of the holy river Ganges (Ganga), very close to Triveni Sangam, i.e., the confluence of three sacred rivers: Ganges, Yamuna (Jamuna) and the mythical Saraswati. The sacred tree, which belongs to the Muslim community, is positioned at only 5 m from the “maazar” (tomb) of the Sufi Saint Saiyid Shah Sadrul Haq Taqiuddin (1320–1384), popularly called Baba Shaik Taqi ,24. It iThe GPS coordinates are 25°25.431’ N, 081°53.981’ E and the altitude is 99 m. The average annual rainfall in the area is 981 mm (Allahabad station) and the mean temperature reaches 25.7°C, with 5–10 frosty days per year .The massive baobab grows on a slope, which tends to widen due to continued errosion by floods of Ganges and water flushing down from adjacent higher areas. The tree was severely damaged during the Kumbh Mela event in 2013, when its eastern side was accidentally set on fire by pilgrims who camped close to the baobab .The maximum height of the tree is h = 14.0 m. The trunk, with the shape of a haystack, forks at heights of 5.5–7.1 m into 6 primary branches, out of which 3 develop horizontally; at least 4 primary branches are missing. The primary branches have diameters between 0.6–1.2 m. The current circumference of the trunk (at 1.30 m above mean ground level) is cbh = 18.27 m, with two severely damaged and broken stems. We estimate the restored girth, before the fire of 2013, to a value cbh = 21.20 m.3. The baobab still produces dozens of pods.According to our visual inspection, the baobab exhibits a cluster structure and consists of 7 fused stems, out of which 6 are old. The horizontal dimensions of the large hemispherical canopy are 35.9 (NS) x 24.1 (WE) m. The total wood volume of the tree is V = 130 mThe baobab has many hollow parts in the stems and branches. Two relatively large irregular cavities can be observed inside the two damaged stems. It also has 3 big roots, partially exposed due to errosion, which spread along the ground up to 30 m. Some of the bark has been extracted, stripped, sliced and peeled off by locals for medicinal use, especially for treating malaria, cold, flu and diarrhea. These actions exposed the soft tissues to fungal and bacterial attacks.At the beginning of 2019, the area around the sacred baobab was filled with mud and sand and a concrete boundary wall was erected around the tree for preventing further errosion. The concrete wall has many openings for excess water drain off .The Parijaat tree at Kintoor, which is worshipped by the Hindu community, has an ancient background. There are many legends around this tree, which might be the most sacred of India. The Parijaat (or Parijat) tree is considered to be a “Kalpavriska” or wish bearing tree, which is only found in heaven. It is claimed that this tree, located near a temple established by Kunti, grows from Kunti’s ashes. Kunti was the mother of the three Pandava brothers, including Arjuna. Arjuna is the main hero of Mahabharata and plays a central role in Bhagavad Gita. According to another saying, Arjuna brought this tree from heaven and his mother Kunti used to crown Lord Shiva with its flowers. The most widespread legend in the area states that Lord Krishna himself brought this tree from heaven, more than 5,000 years ago, for his beloved queen Rukmini.For a long time, this tree was considered to be the only one of its kind. However, in 1971, Maheshwari identified the Parijaat tree to be an African baobab. He measured its girth to 13 m and estimated its age to be anything from 600 to 5000 years .The Parijaat tree is located at 8 km south of the village of Kintoor (or Kintur) and at 38 km north-east of the Barabanki city, in the Barabanki district. The GPS coordinates are 27°00.206’ N, 081°28.922’ E and the altitude is 106 m. The average annual rainfall in the area is 941 mm (Barabanki station) and the mean temperature reaches 25.6°C, with around 5 frosty days per year.The tree is situated in a closed complex, which is a place of much religious significance for the locals. The baobab is surrounded by two metal fences. There is a small temple dedicated to Krishna at its base. According to orders of the district magistrate, the temple was fenced off and is not accessible any more .The maximum height of this tree is h = 13.7 m. The short trunk is of conical shape. It forks at heights of 4.0–5.0 m into 7 very large primary branches, some of them broken, with diameters between 1.2–2.0 m. At least 3 primary branches are missing. The branch sizes are exaggerated in comparison with the relatively modest trunk size . The cir3, out of which 50 m3 belongs to the trunk and 45 m3 to the canopy. According to locals, over the past century at least, the tree did not produce pods.The Parijaat tree also exhibits a cluster structure and consists of 4 or 5 perfectly fused stems. The horizontal dimensions of the very impressive canopy are 33.4 (NS) x 31.6 (WE) m. The overall wood volume is of 95 m−3 m long , were extracted from predetermined positions/distances along every sample. Additionally, we collected with a sharp instrument three tiny samples from one severely damaged stem of each baobab. These additional samples were labelled JA-2, JA-3, JA-4, PK-2, PK-3 and PK-4.As agreed with the local religious leaders, one sample was collected with the increment borer  from one presumptive old stem of each baobab. These two samples were labelled JA-1 and PK-1 . Several2, which was next reduced to graphite on iron catalyst [The α-cellulose pretreatment method was used for removing soluble and mobile organic components . The rescatalyst ,27. The The AMS radiocarbon measurements were done at the AMS Facility of the iThemba LABS, Johannesburg, Gauteng, South Africa, using the 6 MV Tandem AMS system . The obtRadiocarbon dates were calibrated and converted into calendar ages with the OxCal v4.3 for Windows , by usinThe water content of one stem of each baobab was determined by dehydration of wood segments (depth 0.20–0.30 m) for 72 h, at 120°C, under ambient atmosphere.Radiocarbon dates, expressed in radiocarbon years BP  and calibrated ages  of 11 sample segments are listed in For three sample segments with lower positive radiocarbon dates , the 1σ distribution corresponds to several ranges which have all very low probabilities. In these cases, we used for calibration the higher 2σ probability distribution (95.4%), which corresponds to two or three age ranges. We used the same approach for selecting the cal CE range of each sample segment, with one exception. This exception is the segment JA-1b, for which we selected the range with the second highest probability, which agrees better with the age sequence along the sample JA-1.In all cases, the selected age range is marked in bold in For three sample segments , ages fall after 1950 CE (0 BP), namely the 14C activity, expressed by the ratio 14C/12C, shows higher values than the standard activity registered in the reference year 1950. These results correspond to negative radiocarbon dates and are named greater than Modern (>Modern). Such cases indicate a very young age of the dated wood, which was formed after 1950 CE.Sample ages represent the difference between the year 2019 CE and the mean value of the selected age range (marked in bold). The sample ages and the corresponding errors, which are expressed in calendar years, were rounded to the nearest 5 years.We used this approach for selecting calibrated age ranges and single values for sample ages in all our previous articles on AMS radiocarbon dating of large and old angiosperm trees, especially of baobabs ,13,14,22The two samples collected with the increment borer were relatively short, i.e., 0.31 m for JA-1 and 0.40 m for PK-1, even if the penetration of the borer was almost complete. This reveals that both trees have large hollow parts in the corresponding stems. The radiocarbon date of the deepest segment was 188 ± 30 BP for JA-1c and 507 ± 29 BP for PK-1b. These values correspond to calibrated ages of 250 ± 45 and 595 ± 10 yr.For the baobab of Jhunsi, Allahabad, the oldest dated sample JA-4 was collected close to the pith of a stem severely damaged by the fire of 2013. The radiocarbon date of 779 ± 41 BP corresponds to a calibrated age of 770 ± 25 yr. In the case of the Parijaat tree at Kintoor, the oldest dated samples PK-2 and PK-4 were also collected close to the pith of a stem, which broke off several decades ago. Their radiocarbon dates of 793 ± 37 BP and 693 ± 36 BP correspond to calibrated ages of 775 ± 25 and 735 ± 15 cal yr.The very young ages of three samples , with negative radiocarbon dates, collected from areas close to those of the oldest samples, can be explained by the presence of young regrowth wood for healing produced just after the major damage suffered by the respective stems .The radiocarbon dates and calibrated ages of the oldest dated samples  suggest ages close to 800 yr for the stems from which they originate. Because both baobabs have a cluster structure and consist each of several fused stems with close ages, we consider that the age of the baobab of Jhunsi Allahabad, as well the age of the Parijaat tree at Kintoor, is between 750–850 yr, namely 800 ± 50 yr, which is considerably older than we have expected. One can state that both baobabs started growing around the year 1200 CE.By these values, both trees become not only the oldest African baobabs from India, but also the oldest baobabs outside Africa with accurate dating results. The former record holder was the Big Biobab of Mannar Town (Sri Lanka), which is around 750 yr old .The baobab wood has usually a high water content, up to 79%. Big baobabs stay erect/upright mainly due to the weight of stems. When this weight drops to a critical level due to water loss, the stability of the baobab is affected ,14.We measured the water content of the stems, which were sampled with the increment borer. We found extremely low values, more precisely 45.2% for the baobab of Jhunsi, Allahabad and only 39.7% for the Parijaat tree at Kintoor. This low values indicate that both baobabs are close to the end of their life cycle and may topple in a near future.The research reports the results of the AMS radiocarbon investigation of two sacred African baobabs from the Uttar Pradesh state, in northern India. The two investigated trees are the baobab of Jhunsi, Allahabad and the Parijaat tree at Kintoor. Both baobabs have a cluster structure and consist of several fused stems. Several wood samples were collected from the exterior of stems, as well as from deep areas of severely damaged stems. The oldest samples have radiocarbon dates of 779 ± 41 BP for the baobab of Jhunsi and 793 ± 37 BP for that of Kintoor. These dates correspond to calibrated ages of 770 ± 25 and 775 ± 25 yr. According to these values, the age of both baobabs is close to 800 yr. Thus, the baobab of Jhunsi, Allahabad and the Parijaat tree at Kintoor become the oldest dated African baobabs outside Africa.The general state of deterioration and the low water content of stems indicate that the two sacred baobabs are in decline, close to the end of their life cycle.


Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development. OCLN) gene cause a recessively inherited severe human disorder of microcephaly and band-like calcifications with polymicrogyria (BLC-PMG) characterized by loss of cortical convolutions, shallow or absent sulci, and multiple small gyri giving the cortex surface a roughened irregular appearance  existed in the VZ before and after the NE-to-RGC transition. At E10.5, mOCLN is localized to the neuroepithelial plasma membranes and at the centrosome during interphase (Ocln transcripts are annotated in the Ensembl database: (1) full-length transcripts with varying 3’ and 5’UTR lengths, all encoding wild-type OCLN (mOCLN-FL) with four transmembrane domains, two extracellular loops with intracellular N- and C-termini, and (2) one truncated transcript (ENSMUST00000159459) lacking exons 2 and 3, resulting in the loss of the N-terminus and three of the four transmembrane domains   junctions  prior toterphase  and mitoterphase . By E14.terphase . Surpristerphase . Several E0CZ73) . Centros E0CZ73) . In contOcln loss-of-function mouse line was previously generated by excising exon three and was characterized as a homozygous null model  at E10.5 and E14.5 in the developing cortex  compared to wild-type  division outcomes, leading to more apoptotic events and early cell cycle exit, generating neurons at the expense of the progenitor pool . Thus, w M-phase . We obseild-type , consistild-type , we obseild-type . The incant mice , indicatant mice .A second recurring mechanism in mouse models of microcephaly is premature detachment of RGCs from the ventricular surface as a result of centriole duplication impairments and subsequent loss of centrosome and cilia . The attOcln-mutants. Similar to OCLN knockdown in MDCK cells that impaired mitotic cleavage angle of those dividing kidney epithelial cells , neurons, and/or apoptotic events . Both E1al cells , we alsoal cells  in mutanal cells . Taken tOcln-mutant cortices  in humans limits the ability of mouse models to recapitulate all aspects of human brain malformations . Human ent mouse  and the patients . The humand sg11 . Six humand sg11 , compareand sg11 . A list SC lines . Thus, oAs in the mouse, full-length hOCLN (hOCLN-wt) can be found in both membrane and soluble nuclear fractions . EndogenOCLN, we generated 3D cortex-like organoids  from our control and gene-edited hESC lines using a modification of an established protocol  genome-edited hESC lines  and the colony was bred and maintained according to protocols approved by the Institutional Animal Care and Use Committee of Weill Cornell Medical College. Since homozygous mutants were reported to have difficulties breeding  was maintained in feeder-free conditions on vitronectin-coated plates in mTESR1 maintenance media . Cells were routinely passaged with ReLeSR (StemCell Technologies) according to manufacturer’s instructions. Chromosome integrity of hESCs was confirmed by G-banding karyotype analyses performed in the pathology laboratory at Weill Cornell Medical College. Mycoplasma testing was carried out using a MycoAlert kit (Lonza). Briefly, cells at 70% confluency were washed once with 1x PBS, incubated with ReLESR for 1 min at 25°C, and once ReLESR was removed, the cells were incubated dry for 3 min at 37°C. Cells were then resuspended in warm mTESR1 media and transferred to freshly-coated plate at a dilution of 1:10 to 1:15.http://chopchop.cbu.uib.no/ and were cloned into pSpCas9(BB)−2A-Puro vector . pSpCas9(BB)−2A-Puro vector (see Key Resource Table) was transfected in H9 hESCs plated at 50–70% confluency, using Amaxa Human Stem Cell Nucleofector kit (Lonza VPH-5002). Cells positive for the pX459 vector were selected for after 48 hr puromycin treatment (0.5 ug/mL) and the surviving cells were re-seeded in 96-well plates at clonal dilution and wells containing single clones were marked. After 7 days of culture, clonal colonies were harvested, gDNA was extracted, and PCR-amplified using primers found in to validate homozygous mutation  containing DMEM/F-12, 1X B-27 serum without Vitamin A, 1X N2 supplement, 1X GlutaMax, 1X MEM Non-essential Amino Acid, 100 U/ml penicillin-streptomycin, and 2-mercaptoethanol. For neural induction, the media was supplemented with SB-431542 and LDN193189 for the first 5 days of culture (d0-d5). In addition, to promote single-cell survival, medium was the medium was supplemented with 10 μM ROCK inhibitor Y-27632 for the first 24 hr (d0). On d6, floating organoids were transferred to ultra-low attachment six-well plates in FNIM supplemented with 20 ng/mL bFGF and 20 ng/mL EGF, with daily media changes from d6-16 and media changes every other day from d17 to d25. On d25, media was replaced with Cortical Spheroid Media (CSM) containing Neurobasal  were generated according to a previously published protocol  with sevE10.5-E16.5 embryos were drop-fixed in 4% PFA in PBS at 4°C for 12–16 hr, cryoprotected in 15% then 30% sucrose for a total of 48 hr, then embedded in OCT and flash-frozen. Each block was cryosectioned at −20°C at 10–14 μm, depending on embryonic age, and was stored at −20°C until ready to be immunostained. P1, P7, and P20 pups were perfused with PBS followed by 4% PFA in PBS. Brains were harvested, drop-fixed in 4% PFA in PBS for 12 hr at 4°C, and cryoprotected as above.Cortical organoids  were washed once with PBS, fixed in 4% PFA in PBS at 4°C for 12–16 hr, and cryoprotected in 30% sucrose for 48 hr. They were embedded in OCT in Tissue-Tek Cryomolds (10 × 10×5 mm) and flash-frozen (about 50 organoids were embedded per mold). Each frozen mold was cryosectioned at 10 μm and sections were stored at −20°C until immunostained.Frozen sections were washed in PBS three times for 5 min each and were heated at 95°C for 15 min in antigen-retrieval buffer. They were then blocked for 1 hr with 10% Normal Donkey Serum (NDS) in PBS-Tween (0.1% tween) and incubated in primary antibody (see Key Resource Table) in blocking buffer at 4°C for 16–20 hr. Sections were washed with PBS (3 × 10 min) and incubated in secondary antibody in blocking buffer at room temperature for 1 hr. Sections were washed with PBS (3 × 10 min), incubated with DAPI for 10 min, and mounted using Prolong Antifade, based on manufacturer’s instructions.Immunocytochemistry hESCs were seeded on vitronectin-coated cover slips (Neuvitro #GG-18-pre) and were cultured until 50% confluent. Cells were washed once with PBS and fixed with 4% PFA in PBS for 20 min at room temperature. They were washed twice with PBS and heated at 95°C for 10 min in antigen-retrieval buffer. They were then blocked for 1 hr with 10% NDS in PBS-Tween (0.1% tween) and incubated in primary antibody (Key Resource Table) in blocking buffer at 4°C for 16–20 hr. Cover slips were washed with PBS (3 × 10 min) and incubated in secondary antibody in blocking buffer at room temperature for 1 hr. Cover slips were washed with PBS (3 × 10 min), incubated with DAPI for 10 min, and mounted using Prolong Antifade, based on manufacturer’s instructions.Key Resource Table). Adherent hESCs were manually harvested using cell scraper at 70% confluency, centrifuged at 500 x g for 5 min, and washed once with ice-cold PBS. The pellet was resuspended in ice-cold cytoplasmic extraction buffer (CEB) supplemented with 1x Protease inhibitor cocktail and centrifuged at 500 x g for 10 min. The supernatant (cytoplasmic extract) was collected into a clean tube, flash frozen and stored at −80°C. The remaining pellet was resuspended in ice-cold membrane extraction buffer (MEB) with 1x protease inhibitor, quickly vortexed, incubated at 4°C for 10 min, and centrifuged at 3000 x g for 5 min. The supernatant (membrane extract) was collected into clean tube, flash frozen, and stored at −80°C. The remaining pellet was resuspended in ice-cold nuclear extraction buffer (NEB) with 1x protease inhibitor, vortexed for 15 s, incubated at 4°C for 30 min, and centrifuged at 5000 x g for 5 min. The supernatant (soluble nuclear extract) was collected into clean tube, flash frozen, and stored at −80°C. The remaining pellet was resuspended in room-temperature NEB with 5 mM CaCl2, three units/uL of micrococcal nuclease, and 1x protease inhibitor, vortexed for 15 s, incubated at 25°C for 15 min, and centrifuged at 16,000 x g for 5 min. The supernatant (chromatin-bound extract) was collected into clean tube, flash frozen, and stored at −80°C. Fractionation of mouse tissues were performed as described above, but were first homogenized using a Polytron handheld homogenizer.Fractionation was performed using Thermo Fisher’s kit for cultured cells and tissue following manufacturer’s instructions (RNA was isolated from 3-D spheroids or from E12.5 mouse embryos using TRI reagent (Sigma T9424) per manufacturer’s instructions. Briefly, cells or mouse tissue was homogenized, resuspended in 1 mL TRI reagent, and incubated at 25°C for 5 min. 100 uL of 1-bromo-3-chloropentane was added for phase separation and after rigorous mixing and 10 min incubation at room temperature, the sample was centrifuged at 12,000 x g for 15 min at 4°C. The upper aqueous layer was transferred to a fresh tube and mixed with 500 μL of 100% isopropanol and centrifuged at 12,000 x g for 10 min at 4°C. Supernatant was discarded and the remaining pellet was resuspended in 75% ethanol in DEPC-treated water, centrifuged at 7500 x g for 5 min at 4°C. The RNA pellet was air-dried for 10–20 min, resuspended in DEPC-treated water, and flash-frozen for long-term storage at −80°C. Purified RNA was reverse transcribed using the iScript cDNA kit and PCR was performed using primers in Mouse tissue or hESC were lysed in ice-cold RIPA buffer supplemented with 1x Protease and Phosphatase inhibitors. The concentration of the protein lysate was determined using BCA Protein Assay Reagent. 30–40 micrograms of protein were separated on a 4–12% NuPage Bis-Tris protein gels and transferred to 0.2 μm nitrocellulose membranes (Biorad). The membranes were blocked in Odyssey Blocking Buffer at 25°C for 1 hr, incubated with primary antibodies at 4°C overnight, washed three times with TBS, incubated with secondary antibodies for 1 hr, and imaged using Odyssey Imager. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study convincingly characterizes a previously unrecognized function for the tight junction protein Occludin (OCLN) at centrosomes/mitotic spindles to regulate the neuroepithelial to radial glial cell transition in vivo in the mouse and in human ESC-derived cortical organoids. Previous work reported localization of OCLN to centrosomes and regulation of mitosis. A seminal finding of this study is the demonstration of the expression characteristics of full length (FL) OCLN and a truncated form lacking the portion encoded by exons 2 and 3. It is the loss of OCLN-FL that impairs cortical development. Presumably via its interaction with the mitotic spindle protein NuMa the investigators nicely show that OCLN functions to stabilize mitotic spindles and thereby impacts M-phase length, apoptosis, and importantly the proper balance between neural progenitor pool size and differentiated neurons.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Sarah Lutz (Reviewer #2), Leslie M Thompson (Reviewer #3).Thank you for submitting your article "Tight junction protein occludin regulates progenitor self-renewal and survival in developing cortex" for consideration by The reviewers discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The results reported in this manuscript provide novel fundamental biological insight regarding the function of the protein OCLN that will be of broad interest for a readership focused on neurodevelopment. Mutations in OCLN, previously believed to be exclusively a tight-junction protein, result in microcephaly and cortical malformation. This study convincingly characterizes a previously unrecognized function for the tight junction protein Occludin (OCLN) at centrosomes/mitotic spindles to regulate the neuroepithelial to radial glial cell transition in vivo in the mouse and in human ESC-derived cortical organoids.Essential revisions:In Figure 2F – legend states that T-test was done. It is suggested that authors use ANOVA with posthoc for statistical analysis of multiple and timepoints groups.Minor points:1) Figure 3F, the X axis should go from 0-100 and Figure 1A "enlarged" lacks a scale bar.2) The Discussion could be more focused on the major contribution of this study, i.e. loss of OCLN-FL that impairs cortical development. In this light, what is known about the human OCLN mutations that cause microcephaly – are they consistent with the importance of OCLN-FL in cortical development? In addition, less space in the Discussion should be devoted to aspects of OCLN in tight junctions and role of truncated OCLN, points that are not addressed by data in this manuscript.3) The authors interpret their data to suggest that the phenotypes they observe are due to loss-of-function caused by loss of full length OCLN. Could an alternative explanation be that some or all of those phenotypes are caused by or contributed to by gain-of-function of OCLN-ΔN?Ocln mutant mice did not exhibit gross phenotypes that would be expected of impaired TJs" to mean that "OCLN plays no part in TJ maintenance or formation". Could an alternative explanation be that OCLN-ΔN is insufficient for TJ formation and maintenance? In general, it would be helpful for the authors to comment specifically about truncated forms of OCLN in both mouse and human and their roles. Especially since it is shown to be mainly nuclear localized and specifically localized at centrosomes. The authors make a strong case for the full-length protein but a comment in the Discussion about the truncated version and its roles would be interesting to readers particularly if there are thoughts on whether full length is playing similar role at centrosome as truncated.4) The authors interpret previous results showing that "5) Some OCLN staining presented throughout the manuscript was interpreted to be localization to the plasma membrane, but appears to also localize to the cytosol. For instance, in Figure 2C there appears to be some diffuse nuclear staining of n-term OCLN in the ΔN/ΔN mice strains. This is visible in all cells from 10.5 to 12.5. Additionally, OCLN is a well-known tight junction protein, but does not appear to show such localization – is this a temporal effect during development?6) Some figure legends are missing information such as how many mice per genotype were assessed per figure. What do the red arrows show in Figure 3E? There doesn't appear to be DAPI staining in Figure 4A, but it is listed in the legend. In Figure 6B, please indicate how many spheroids were analyzed, how many experiments were performed, and what the error bars represent. In Figure 6G, what do the error bars represent? In Figure 7, please indicate antibody as it was shown to be important in previous figures.7) In the Abstract there is little mention of temporal expression differences of OCLN isoforms. This is an interesting finding from this manuscript that isn't highlighted until the final sentence.8) Gene and protein names don't always appear to be written appropriately for the species being presented/discussed. A few examples include OCLN when presenting mouse data.9) Figure 1H should have the axis range from 0-1.45. Rationale is that the magnitude of the reduction in cortical size throughout the paper is small – which is fine, it is small but carefully characterized and phenotypically important.10) Please provide double immunostaining of full length and truncated OCLN and a vascular marker. This could be done with existing mouse brain sections such as those used in figures looking at a section of cortex, etc.eLife.Congratulations, we are pleased to inform you that your article, "Tight junction protein occludin regulates progenitor self-renewal and survival in developing cortex", has been provisionally accepted for publication in The manuscript has been improved but there are some remaining issues that need to be addressed before publication, as outlined below:1) For the following response to review:In the authors’ response to point 5: “While it is known that OCLN is transported to and from various subcellular locations as part of normal intracellular cell processing, reports-including IP-mass spectrometry proteomic screens  have failed to detect an intranuclear function of OCLN . This diffuse signal the reviewer refers to is likely background, non-specific antibody labeling accentuated at higher magnifications that focus on a single cell. Indeed, in the lower magnification images in Figure 1, the nuclei appear as voids in the OCLN antibody stains.”Upon cursory examination of Fredriksson et al., 2015 (assuming the correct publication was found), IP-MS does not appear to have been performed on the mouse model used here (ΔN/ΔN OCLN), preventing correlation of the findings in their report with those of this manuscript. The authors imply that the nuclear signal observed is accentuated at the higher magnification, however clear nuclear signal is present even at lower magnification, specifically in ΔN/ΔN animals at E10.5 . Without proper controls , the response that the nuclear signal  is "likely background" cannot actually be verified. Additionally, the statement that nuclei void of OCLN staining in Figure 1 supports these claims cannot be verified – Figure 1 states that mAb and pAb against OCLN were used while Figure 2C states that an N-term OCLN antibody was used. With the information given, reviewers are unable to verify whether the same antibody was used in these figures. While additional staining would clarify, we would only request that a revision of the text to support perhaps unknown and undescribed nuclear function for OCLN in ΔN/ΔN animals.2) In the authors’ response to point 8: “Care has been taken to ensure that OCLN is appropriately referring to encoded protein in all instances in the manuscript.”Some figures need edits  to capitalize proteins, for consistency.One last thing – in their list of figure legends they listed "Source Data" for certain figures. They took out a lot of the experimental details (numbers of mice etc.) and indicated in the rebuttal that it was included. We are assuming it is in source data, but we did not see those files. Essential revisions:In Figure 2F – legend states that T-test was done. It is suggested that authors use ANOVA with posthoc for statistical analysis of multiple and timepoints groups.The authors have changed the statistical analysis performed for Figure 2F to two-way ANOVA with Sidak’s multiple comparison test, comparing WT and mutant within each age group.Minor points:1) Figure 3F, the X axis should go from 0-100 and Figure 1A "enlarged" lacks a scale bar.The x-axis for Figure 3F has been updated to match reviewers’ comments. A scale bar has been added to the “enlarged” image in Figure 1A; it represents 25 microns, which is the same length represented by all other scale bars in Figure 1A.2) The Discussion could be more focused on the major contribution of this study, i.e. loss of OCLN-FL that impairs cortical development. In this light, what is known about the human OCLN mutations that cause microcephaly – are they consistent with the importance of OCLN-FL in cortical development? In addition, less space in the Discussion should be devoted to aspects of OCLN in tight junctions and role of truncated OCLN, points that are not addressed by data in this manuscript.The authors agree that a more extensive discussion of the clinical importance of OCLN-FL is necessary – we have updated the discussion accordingly and have shortened the discussion regarding OCLN in the context of tight junctions.3) The authors interpret their data to suggest that the phenotypes they observe are due to loss-of-function caused by loss of full length OCLN. Could an alternative explanation be that some or all of those phenotypes are caused by or contributed to by gain-of-function of OCLN-ΔN?OCLN mutations causing BLC-PMG were found on exons 5, 6, or 7 that would result in loss-of-function of all OCLN isoforms. Third, there is no indication yet to suggest a genotype-phenotype correlation with clinical severity between the more proximal vs. distal OCLN mutations. Together these features strongly indicate that loss-of-function of full-length OCLN underlies the phenotypes observed in this study.While the observed phenotypes could be caused, in part, by a dominant negative effect of the truncated protein (OCLN-ΔN) rather than loss-of-function of full-length OCLN, this is unlikely for several reasons. First, this human disorder is associated in half of reported cases with autosomal recessively inherited, truncating mutations in exon 3, which speaks against a dominant negative effect of the truncated isoform, since heterozygous individuals are asymptomatic. Second, a number of reported insertion/deletion The Discussion section has been updated to clarify this point.4) The authors interpret previous results showing that "Ocln mutant mice did not exhibit gross phenotypes that would be expected of impaired TJs" to mean that "OCLN plays no part in TJ maintenance or formation". Could an alternative explanation be that OCLN-ΔN is insufficient for TJ formation and maintenance? In general, it would be helpful for the authors to comment specifically about truncated forms of OCLN in both mouse and human and their roles. Especially since it is shown to be mainly nuclear localized and specifically localized at centrosomes. The authors make a strong case for the full-length protein but a comment in the Discussion about the truncated version and its roles would be interesting to readers particularly if there are thoughts on whether full length is playing similar role at centrosome as truncated.Ocln mouse mutant, a closer look at the role that this truncated isoform may play in TJ formation is warranted. The Discussion has been updated to clarify this point.The authors did not intend to imply that OCLN plays no part in TJ maintenance or formation. In light of the observations that the OCLN-ΔN is continually expressed in the Very little is known regarding the function of OCLN-ΔN and other shorter isoforms. The data and results analyzed in this study revolve around the role of full-length OCLN. Based on recent reports of astral microtubule function and the astral microtubule disruption demonstrated here in cells lacking full-length OCLN, the authors suggest that this isoform is required for proper mitotic centrosomal positioning to ensure a near perpendicular cleavage plane of symmetrically dividing radial glial cells. The authors thought it best to minimize the amount of text dedicated to the presumed roles of truncated isoforms in the absence of data beyond OCLN-interaction with pericentrosomal matrix proteins NuMA and RAN. However, we fully support any future endeavors to tease out the role(s) of these truncated isoforms in neurodevelopment and neurovascular biology.5) Some OCLN staining presented throughout the manuscript was interpreted to be localization to the plasma membrane, but appears to also localize to the cytosol. For instance, in Figure 2C there appears to be some diffuse nuclear staining of n-term OCLN in the ΔN/ΔN mice stains. This is visible in all cells from 10.5 to 12.5. Additionally, OCLN is a well-known tight junction protein, but does not appear to show such localization – is this a temporal effect during development?While it is known that OCLN is transported to and from various subcellular locations as part of normal intracellular cell processing, reports – including IP-mass spectrometry proteomic screens  have failed to detect an intranuclear function of OCLN . This diffuse signal the reviewer refers to is likely background, non-specific antibody labeling accentuated at higher magnifications that focus on a single cell. Indeed, in the lower magnification images in Figure 1, the nuclei appear as voids in the OCLN antibody stains.6) Some figure legends are missing information such as how many mice per genotype were assessed per figure. What do the red arrows show in Figure 3E? There doesn't appear to be DAPI staining in Figure 4A, but it is listed in the legend. In Figure 6B, please indicate how many spheroids were analyzed, how many experiments were performed, and what the error bars represent. In Figure 6G, what do the error bars represent? In Figure 7, please indicate antibody as it was shown to be important in previous figures.The authors have included the missing information requested by reviewers. Specifically, we have specified how many mice were analyzed in Figure 4D, we have specified what the red arrows signify in Figure 3E, we have fixed the figure legend for Figures 4A and 4C taking into account lack of DAPI presence, we have specified the number of organoids analyzed per group in Figure 6B, and we have specified what error bars represent in Figures 6B and 6G. All antibodies used can be found in the Key Resource Table.7) In the Abstract there is little mention of temporal expression differences of OCLN isoforms. This is an interesting finding from this manuscript that isn't highlighted until the final sentence.The authors agree that this is an interesting and important aspect of OCLN isoform developmental regulation. It was not emphasized in the Abstract due to word limitations. The Abstract has been revised to highlight this feature while streamlining the text to maintain the 150 word limit.8) Gene and protein names don't always appear to be written appropriately for the species being presented/discussed. A few examples include OCLN when presenting mouse data.https://www.biosciencewriters.com/Guidelines-for-Formatting-Gene-and-Protein-Names.aspx]. Care has been taken to ensure that OCLN is appropriately referring to encoded protein in all instances in the manuscript.The accepted convention for nomenclature is to italicize in all capital letters to indicate human genes while only the first letter is capitalized for mouse genes. However, the encoded proteins should be in all caps, no italics, for human and mouse proteins. The manuscript has been improved but there are some remaining issues that need to be addressed before publication, as outlined below:1) For the following response to review:In the authors’ response to point 5: “While it is known that OCLN is transported to and from various subcellular locations as part of normal intracellular cell processing, reports-including IP-mass spectrometry proteomic screens  have failed to detect an intranuclear function of OCLN . This diffuse signal the reviewer refers to is likely background, non-specific antibody labeling accentuated at higher magnifications that focus on a single cell.Indeed, in the lower magnification images in Figure 1, the nuclei appear as voids in the OCLN antibody stains.”Upon cursory examination of Fredriksson et al., 2015 (assuming the correct publication was found), IP-MS does not appear to have been performed on the mouse model used here (ΔN/ΔN OCLN), preventing correlation of the findings in their report with those of this manuscript. The authors imply that the nuclear signal observed is accentuated at the higher magnification, however clear nuclear signal is present even at lower magnification, specifically in ΔN/ΔN animals at E10.5 . Without proper controls , the response that the nuclear signal  is "likely background" cannot actually be verified. Additionally, the statement that nuclei void of OCLN staining in Figure 1 supports these claims cannot be verified – Figure 1 states that mAb and pAb against OCLN were used while Figure 2C states that an N-term OCLN antibody was used. With the information given, reviewers are unable to verify whether the same antibody was used in these figures. While additional staining would clarify, we would only request that a revision of the text to support perhaps unknown and undescribed nuclear function for OCLN in ΔN/ΔN animals.The reviewer makes an excellent point, and we have accordingly modified the Results section to comment that diffuse OCLN nuclear labeling was detected in VZ cells. We add in the Discussion that we cannot with our present data rule out the possibility of a nuclear role for one or more OCLN isoform and note that OCLN has been found in a previously reported proteomic screen to associate with a nuclear envelope protein, ezrin.2) In the authors’ response to point 8: Care has been taken to ensure that OCLN is appropriately referring to encoded protein in all instances in the manuscript.Some figures need edits  to capitalize proteins, for consistency.We have modified the figures to ensure that labeling of the panels consistently use all capitals for protein abbreviationsOne last thing – in their list of figure legends they listed "Source Data" for certain figures. They took out a lot of the experimental details (numbers of mice etc) and indicated in the rebuttal that it was included. We are assuming it is in source data, but we did not see those files.Indeed, this information  is included in the source data. We have added this information to the figure legends for ready access.eLife.We thank the reviewers for their many helpful comments that have significantly improved the manuscript. We hope that it is now suitable for final approval and publication in


We aimed to use of IL-12 and IFN-γ for prevention and treatment of hydatidosis in experimental animal models.This experimental study was conducted in Ahvaz City, southwest of Iran in 2017. Forty female BALB/C mice with 6–8 wk old were divided into four groups (two group test and two group control). The mice were challenged with 2000 protoscolices and a combination of IL-12 (10 μg/kg) and IFN-γ (25 μg/kg) was injected intraperitoneally daily one week at the time of challenging (prevention group) and three months after challenging (treatment group). After four months, the mice were euthanized and the number, size, and weight of the cysts were measured in all studied groups. Pathologic slides were prepared from cyst wall for pathologic changes study.There issignificant reduction in number, size, and weight of cysts in test groups.Cytokine therapy is effective methods especially for prevention of cyst formation and to treat non-operable cases. Further study of combination of chemotherapy and cytokine therapy should be investigated. Echinococcus granulosus a cestode worm which located in small intestinal of dogs. The disease causes economic losses in animal husbandry field and costs spending in human population for surgery and other medical procedures. The distribution of the disease is worldwide especially in area with poor sanitation, poor living condition and more closer to dogs and sheep  acquired infection via ingestion of eggs which passes from feces of canine family (definitive host). Hydatid cyst is a form of larva stage of nd sheep , 2.Treatment of hydatid cyst in animal husbandry is not common but in human, there are many treatment approaches. Surgery is the first choice to remove the cyst in operative cases. The surgery method has some limitation for example in patients who cannot or do not want to undergo surgery, spillage of viable protoscolices and recurrence of infection with forming secondary cyst, patients with several cysts in different organs, in no operative cases due to inaccessibility of the cyst and infections after operation .AlbendazAccording to immune responses to hydatid cyst, there are four type of hydatidosis by the use of ultrasonographic finding which consists type I simple cyst with no internal architecture, type II cyst with daughter cyst at periphery, type III calcified cyst, type IV complicated cyst (ruptured cyst) . VarietyIL12 and IFN-γ are two major cytokine of Th1 axis. IL-12 is a heterodimeric cytokine which produced by variety of immune cells like antigen presenting cells, macrophage and lymphocytes , 10. IL-IFN-γ is a heterodimeric cytokine and is the only member of type II class of interferons . The rolWe evaluated the therapeutic effect of Th1 cytokines (IFN-γ and IL12) on hydatidosis in experimental animal model.Forty female BALB/c mice with 6–8 wk old were obtained from Laboratory Animals Breeding Center of Ahvaz Jundishapur University of Medical Sciences in 2017. Mice were divided into four groups contained 2 group control and two groups for treatment and prophylaxis assay.All animal right ethics principles were considered according to animal right committee protocols and the study was approved by Ethics Committee of the university.Hydatid cysts were collected from infected organs of sheep in Ahvaz abattoir and transfer to parasitology laboratory of medical school. The cysts were opened under sterile condition and protoscolices were collected in sterile tubes. The protoscolices were rinsed 5 times with RPMI 1640 medium and add penicillin-streptomycin 500 unite/ml for inhibiting bacterial growth. The viability of protoscolices was tested by vital staining like eosin and more than 95% protoscolices viability were accepted for challenging BALB/c mice.Two thousand live protoscolices were injected intraperitoneally in BALB/c mice. In prevention group, after challenging with protoscolices, IL12 25 (10 μg/kg) (R&D systems MA USA)  and inteP.value <0.05 was considered for significant differences. The used software was SPSS Fisher exact test and chi-square were used for statistical analyzing and P<0.05). The results of treatment and control groups also showed significant differences between two groups which the number of cysts were 5.7 and 15 respectively (P<0.01). The results are presented in The average number of cyst in prevention and control groups were 2 and 1551, respectively; there are significant differences between prevention and control group for number of Hydatid cyst (P<0.001)  .P<0.05)  . Fig. 2 Using surgery and chemotherapy methods with benzimidazole family drugs is currently applied for treating human hydatidosis. Some adverse effects of two methods lead us to select the other alternative choice. The advantage of cytokine therapy is confirmed and applied for treatment or prophylaxis of some infectious diseases. The benefit of cytokine therapy in human tuberculosis was shown . The useThe benefit of cytokine therapy in visceral leishmaniasis , 26, astLeishmania major infection in BALB/c mice was presented. The treatment of infected BALB/c mice by IL-12 one week after challenging with L. major showed a significant healing due to promotion of Th1 and suppression of Th2 cytokines . The parasites load in heterozygous mice was intermediate between LT- α TNF- α −/− and wild-type mice . DecreasE. multilocularis-infected mice (C57BL/6 J) was evaluated. The administration of IL-12 at the challenging time reduced parasite burden and inhibit development of disease. The combination of IL-12 and albendazole should be considered for treatment and prevention of alveolar echinococcosis in human (The effect of IL-12 injection to in human .We evaluated the efficacy of cytokine therapy for treatment of progressive hydatidosis condition too. A combination of IL-12 and IFN- γ administration to echinococcosis-infected mice three months after challenging, reduced number, weight and size of cyst 63%, 81.5%, and 18.3% respectively. Although the administration of cytokines after establishing infection significantly was effective, the use of cytokine therapy was more effective for prevention purpose, agreement with Emery study (Using Th1 cytokines could be effective on treatment and prevention of hydatidosis in human especially for prevention of secondary hydatid cyst development. Further studies are needed to evaluate treatment and prevention effect of cytokine and chemotherapy combination for hydatidosis.


In this paper, the fracture behavior of GaN piezoelectric semiconductor ceramics was investigated under combined mechanical and electric loading by using three-point bending tests and numerical analysis. The experimental results demonstrate that, in contrast to traditional insulating piezoelectric ceramics, electric current is a key factor in affecting the fracture characteristics of GaN ceramics. The stress, electric displacement, and electric current intensity factors were numerically calculated and then a set of empirical formulae was obtained. By fitting the experimental data, a fracture criterion under combined mechanical and electrical loading was obtained in the form of an ellipsoid function of intensity factors. Such a fracture criterion can be extended to predict the failure behavior of other piezoelectric semiconductors or devices with a crack, which are useful in their reliability design and applications. Piezoelectric semiconductor ceramics (PSCs) are semiconducting ceramic materials that have piezoelectric properties ,2,3. SinHowever, PSCs, as a typical kind of brittle ceramics, are highly sensitive to internal flaws, such as cracks and cavities ,13. EspeTo develop the fracture criterion of PSCs, many theoretical efforts have been done. Usually, the fracture criterion of insulating dielectric ceramics ,23,24 isThe paper is structured as follows. The experimental procedure is described in N-type semiconductors with a Curie temperature of 265 °C. It is seen from The material that is used in this work is GaN, which is a new kind of electronic ceramics materials with piezoelectric and semiconductor properties. Due to the stable conductivity and excellent functional properties, GaN is a perfect candidate material of electromechanical, high-power and high-frequency devices for operation in extreme environments ,26,27,28l = 40.0 mm, thickness t = 3.0 mm, and width w = 4.0 mm. Specimens were polarized along its thickness direction using a polarization field of 32 kV cm−1 (the coercive field Ec = 8.19 kV cm−1). Here, it is worth noting that polarization along the length direction of a sample needs a higher voltage, which will be done in our next work. The poling temperature and time were 120 °C and 30 min. During the poling process, two poly tetra fluoro ethylene sheets (PTFE) were bond on both surfaces along the thickness direction of a sample with a thickness of PTFE ts = 0.5 mm. Then, silver paste was plated on the up and bottom surfaces of PTFE sheets. An adjustable DC high-voltage (provided by a power supply of 100 kV) was applied to silver wires that were welded on electrodes. All of the polarization jigs were placed in a plexiglass container that was filled with silicone oil (with a relative dielectric constant of 2.73) to prevent high-voltage discharging. Under an applied electric field (32 kV cm−1), carriers in a sample would be redistributed (electrons accumulate the positive pole and holes towards to the negative pole), and a new internal electric field would be generated, which can cause domain switch and finally make the sample reach the saturation polarization strength. After polarizing, the samples were cleaned by an ultrasonic cleaner and stored in a drum wind drying oven at 70 °C for 10 min.In accordance with the three-point bending fracture test standard of fine ceramics , single-a) ranges from 1.43 mm to 2 mm, and at the bottom of a pre-crack, there is an arc with a diameter between 20 μm and 60 μm .During three-point bending fracture tests, the mechanical load Generally, it is difficult to study the fracture behaviors of materials that were subjected to coupled loading. To further clarify the fracture characteristics of PSCs under a combined electrical and mechanical load, we have to resort to numerical analysis, and thus, a numerical model together with finite element analysis was developed.oxy Cartesian coordinate system was set up, with the origin point o at a sample center and the x and y being symmetrical axes, as illustrated in N-type semiconductor property, the linearized equilibrium equations were given by Hu et al. [ijσ are the stress components , iD and iJ are the components of electric displacement vector and electrical current, respectively, q is the elementary charge, and Δn is the variation of carrier density.An u et al. , that isN-type) with its polarization direction along the y-direction is as follows [u is the elastic displacement in the x-direction and v is the elastic displacement in the y-direction, φ is the electric potential. ijc, ije, and ijκ are the elastic, piezoelectric, and dielectric constants. n0 is the initial carrier density, and ijμ and ijd are the electron mobility and diffusion, respectively.The constitutive relation of two-dimensional PSC σBy substituting Equation (2) into Equation (1), the governing equations of PSC can be obtained asVa is the voltage applied at the left end, νrec is the thermal recombination velocity, and nm is the critical electron density that can be expressed asB is the surface barrier of GaN, T is absolute temperature, kB is the Boltzmann constant, h is the Planck constant, and me = 1.82 × 10−31 kg and Nc = 2.23 × 1024 m−3 denotes the effective mass of a conduction band electron and the effective state density of conduction bands, respectively. Vbi is the built-in voltage [Under the experimental conditions see , the bou voltage ,35 that qΦM (4.26 eV) [qχ (4.10 eV) [The barrier height was formed by Schottky contact ,35 and i4.26 eV) , and the4.10 eV) . TherefoAt the three contact points, the boundary conditions are given byjl is the outer normal vector.On the crack faces, the insulating boundary conditions are adopted as (11)σijlσK, the electric displacement intensity factor, DK, and the electric current intensity factor, and JK, for the given sample geometry and loading conditions , as follows:r is the distance from the crack tip in the y-direction.According to Zhao et al. , all theσK0, DK0, and JK0 are the three intensity factors in the case of ND = 1.29 × 1023 m−3, P = 4 N, and Ea = 106 V m−1.For simplification and comparison, these intensity factors are normalized asy-axis, and thus it is perpendicular to the direction of an applied electric field (in the x-direction), which is uncoupled with the stress component xxσ (see Equation (2a)). Therefore, the electric load has no effect on the stress intensity factor. Similarly, due to the polarization direction along the y-axis, the piezoelectric charge moves in the y-axis direction under the applied mechanical load (piezoelectric-semiconductive effect). The electric current density in the x-direction (xJ) and the electric displacement in the x-direction (xD) are not affected by the mechanical load (see As shown in load see .a/w), the the stress intensity factor, σK, the electric displacement intensity factor, DK, and the electric current intensity factor, and JK, conform to a unique fitting function  are the shape factors of a three-point bending specimen, and i = 1, 2, and 3. je are the fitted coefficients, which are corresponding to the stress, electric displacement, and electric current intensity factors, respectively, as summarized in With reference to the standard form (ISO15732:2003) , the fortion see a–c, thatσKC, = 0.205 ± 0.011 MPa m1/2, which is obviously lower than fracture toughness of insulating piezoelectric ceramics (around 1 MPa m1/2) [As shown in Pa m1/2) ,42,43. A4 A m−2, the mean value of the critical stress intensity factor increases from 0.19 to 0.26 MPa m1/2, and thus fracture toughness increases by 36.8%. However, as the applied electric current further increases, fracture toughness decreases. Under an applied electric current, the corresponding fracture toughness distributions can be completely changed (see It is seen from nged see . That isσ,CK = 0.194 MPa m1/2, the critical electric displacement intensity factor DKC, = 3.409 × 1013 C m−3/2, the critical current density intensity factor JKC, = 3.236 × 103 A m2, with the normalized fitting coefficients d = 51.65, f = 51.64, and g = −1.05. The mechanical and electrical fracture toughness of PSC GaN was obtained. Due to the theoretical simplification and scattering of the experimental data, there are differences between the theoretical and experimental results (see Here, it is worth noting that the fracture criterion of an insulating piezoelectric ceramic can be expressed as an ellipse of two independent variables, i.e., the stress and electric field intensity factors ,45. Accoults see . Neverth6 A m−2 (see When compared to purely mechanical loading, there is an obvious discharge near the crack tip under a combined electrical and mechanical load see . This is m−2 see . In caseThe fracture of PSCs under combined mechanical, voltage, and current loading was investigated by using a three-point bending experiment method. The experimental results show that the electric current density is a key factor to the fracture behavior of PSCs, and thus, an electric current density factor should be involved in the fracture criterion. The expressions of stress, electric displacement, and electric current density intensity factors of PSCs were obtained via finite element analysis. On this basis, a fracture criterion under combined electrical and mechanical loading was established by fitting the experimental data.It is shown that, given that the relevant information on sample geometries and material properties such as mechanical and electrical fracture toughness, it is possible to predict the critical loads and vice versa. It is expected that the fracture criterion and the discovery of new material properties can be helpful in greatly improving the reliability and service life of PSCs, and to reduce use-cost, especially for the derating design of GaN electro-mechanical coupling devices. Finally, it is also worth noting that, to clarify electric current-based mechanisms and consider different polarization and electric field directions, further experimental and theoretical works are still needed.


The clinically relevant two-hit mouse model of VILI, which involves the intravenous injection of lipopolysaccharide prior to high tidal volume (HTV)-mechanical ventilation, was used in this study. To investigate the role of COX-2 in the recruitment of VEGF-secreting Ly6Chigh monocytes during VILI, celecoxib, which is a clinical COX-2 inhibitor, was administered 1 h prior to HTV-mechanical ventilation. Pulmonary vascular permeability and leakage, inflammatory leukocyte infiltration, and lung oxygenation levels were measured to assess the severity of VILI. HTV-mechanical ventilation significantly increased the recruitment of COX-2-expressing Ly6Chigh, but not Ly6Clow, monocytes. Celecoxib significantly diminished the recruitment of Ly6Chigh monocytes, attenuated the levels of VEGF and total protein in bronchoalveolar lavage fluid, and restored pulmonary oxygenation during VILI. Our findings demonstrate that COX-2 activity is important in the recruitment of VEGF-secreting Ly6Chigh monocytes, which are involved in VILI pathogenesis, and indicate that the suppression of COX-2 activity might be a useful strategy in mitigating VILI.Mechanical ventilation is usually required for saving lives in critically ill patients; however, it can cause ventilator-induced lung injury (VILI). As VEGF-secreting Ly6C Mechanical ventilation is required for patients that suffer from acute respiratory failure or acute respiratory distress syndrome (ARDS), which can be triggered by direct pulmonary insult and indirect extra-pulmonary insult . HoweverhighCCR2highCX3CR1low monocytes, which express high levels of Ly6C (Ly6Chigh) and C-C chemokine receptor 2 (CCR2high) and low levels of CX3C chemokine receptor 1 (CX3CR1low), are designated as the inflammatory subset, and they are derived from bone marrow macrophage-dendritic precursor cells that migrate to inflammatory sites during injury [lowCCR2lowCX3CR1high monocytes are designated as the resident subset and they can enter tissues, irrespective of inflammation [high subset, to the lung microvasculature, contributes to pulmonary-vascular injury in a murine model of systemic endotoxemia [The inflammatory subsets of monocytes, which are heterogeneous and pluripotent cells, consists of at least two phenotypically distinct cells that can be distinguished by Ly6C expression that serves as a specific marker for the monocytic lineage in mice ,15,16. Lg injury ,15. In cotoxemia ,18.high monocytes are recruited to the lung in VILI, which participate in a decrease of lung compliance [high monocytes through enhancing vascular endothelial growth factor (VEGF) secretion for increasing pulmonary vascular permeability on causing VILI [high monocytes during VILI. Thus, we sought to investigate the functional role and therapeutic potential of COX-2 in VEGF-secreting Ly6Chigh monocytes during VILI.Cyclooxygenases (COXs), including the constitutive COX-1, are expressed in most tissues, and the inducible COX-2 expressed in lung resident inflammatory, endothelial, and epithelial cells are responsible for catalyzing the conversion of arachidonic acid to prostanoids, which are involved in inflammation, vascular homeostasis, and vascular permeability . Recent mpliance ,12. Our ing VILI , indicathigh monocytes in a clinically relevant two-hit VILI mouse model. Moreover, using celecoxib, which is a COX-2-specific inhibitor, we demonstrated that COX-2 activity is important in mediating the recruitment of VEGF-secreting Ly6Chigh monocytes that contribute to the increase of total protein in bronchoalveolar lavage fluid (BALF) during the development of VILI. Thus, the COX-2 blockade may have a therapeutic value in the treatment of VILI.Presently, we explore the kinetic changes of COX-2-expressing Ly6CWhile a previous study has demonstrated that the expression and activity of COX-2 are critically involved in the pathogenesis of VILI , the dethigh monocytes are recruited during VILI [high monocytes were COX-2-positive and they showed high Ly6C expression (Q2). COX-2-expressing Ly6Clow monocytes were COX-2-positive and revealed low Ly6C expression (Q4). A high degree of SSC and high levels of CD11b expression (P4) were used for the gating and analysis of neutrophils with Ly6C and COX-2 expressions. COX-2-expressing neutrophils were COX-2-positive and showed high Ly6C expression (Q2-1). The quantification of time course recruitment of COX-2-expressing Ly6C monocytes and neutrophils during VILI is shown . Ly6Clow monocytes were F4/80-positive and showed low Ly6C expression (P3). high and Ly6Clow monocytes. The number of Ly6Chigh, but not Ly6Clow, monocytes was higher in the HTV-injured lung than in the lungs of control, non-ventilated mice. Celecoxib had no significant effect on the recruitment of Ly6Clow monocytes . This result denotes the importance of COX-2 and VEGF functional axis in Ly6Chigh monocytes during the development of VILI, and it provides the clinical insights of celecoxib on targeting COX-2-expressing and VEGF-secreting Ly6Chigh monocytes for treating VILI in patients.The recruitment of VEGF-secreting Ly6C of VILI . However culture . Howeverhigh, but not Ly6Clow, monocytes; this contributes to the pathogenesis of VILI via enhancing pulmonary-capillary permeability and -vasculature leakage in reducing pulmonary oxygenation. Celecoxib, which is a clinically used COX-2 inhibitor that affects functional COX-2 and VEGF expressions in Ly6Chigh monocytes exhibits therapeutic value in treating and/or preventing VILI in patients.Mechanical ventilation is used as supportive therapy for patients with acute respiratory failure or ARDS. However, due to the morphological variation of patients’ lungs, even when protective ventilation is used, and the uneven overdistention of regional alveoli, which leads to the exacerbation of VILI , suggesthigh monocytes were not significantly elevated after 2 h, increased by 4 h, and then significantly increased at 6 h At 0 and 2 h, there were fewer COX-2-expressing Ly6Chigh monocytes than neutrophils; at 4 h, there were equivalent numbers of COX-2-expressing monocytes and neutrophils; and at 6 h, there were more COX-2-expressing monocytes than neutrophils. This result indicated that the COX-2-expressing neutrophils are the first cells to arrive in the ventilator-injured lung and they may employ paracrine signaling to recruit the wave of COX-2-expressing Ly6Chigh monocytes that further amplify the acute phase of lung-injured cascade. Since the role of neutrophils in the process of lung injury has been well-investigated [high monocytes was therefore focused on the pathogenesis of VILI in this study.Neutrophils and monocytes are the important cell types in inflammation and VILI ,11. In ostigated , the impstigated . As the stigated ,24, the highCCR2highCX3CR1low monocytes have been demonstrated to be recruited into inflamed tissues through the activation of CCR2, which is a receptor of chemokine C-C-chemokine ligand 2 (CCL2) [high monocytes in VILI is unknown. Moreover, how COX-2-activated prostaglandins or other mediators, including cytokines, chemokines, and growth factors, mobilize and activate Ly6Chigh monocytes in the pathogenesis of VILI remains unclear. CCL2 is required in guiding the migration of CCR2-expressing monocytes into inflamed tissues [high monocytes in injured alveoli is suppressed, protecting the mouse from fibrosis [high monocytes that have been reported to express CCR2 [high monocytes. During VILI, COX-2 activation by proinflammatory mediators may stimulate the secretion of CCL2 to govern the lung margination of inflammatory cells, such as Ly6Chigh monocytes. Moreover, these findings contribute therapeutic insights into the inhibitory effect of celecoxib on the IL-1β-COX-2-CCL2 signaling axis, which suppresses the mobilization of Ly6Chigh monocytes, thereby attenuating VILI.Ly6C2 (CCL2) . However tissues , and CCL tissues . Furtherfibrosis . Moreovefibrosis , are assfibrosis . Here, wess CCR2 , resultihigh monocytes enhance VEGF expression for increasing pulmonary vascular permeability, which leads to VILI [high monocytes time-dependently secreted VEGF, and this VEGF secretion was inhibited by celecoxib, indicating that VEGF secretion by Ly6Chigh monocytes during VILI is COX-2-dependent. Furthermore, these results reveal the importance of COX-2 inhibition by clinical COX-2 inhibitors that are used for treating and/or preventing VILI, via the interruption of the COX-2-VEGF functional axis in Ly6Chigh monocytes.Further, we show that the inhibition of COX-2 activity by celecoxib can significantly diminish the VEGF and BALF total protein levels. It is known that there is a high correlation between the expression of COX-2 and VEGF, and that NS-398, a COX-2 inhibitor, inhibits VEGF expression in cancer cells . Additio to VILI . Presenthigh monocytes, but the role of COX-2-expressing neutrophils in VILI cannot be ignored, because COX-2-expressing neutrophils and Ly6Chigh monocytes may sequentially interplay with each other during VILI. A previous study showed that a COX-2 inhibitor potently inhibits the recruitment of neutrophils during VILI [high monocytes in late to prevent the VEGF-stimulated pulmonary permeability increment. A previous study showed that the expression of induced form of COX-2 is relatively low in mouse lung tissue, but it is strongly upregulated after ventilation, especially HTV for 4 h, indicating an acute phase injury of ventilation on upregulating COX-2 expression. Furthermore, the COX-2 expressing cells in ventilation-injured lung were verified using immunohistochemically staining, which show a significant positivity staining of pulmonary alveolar and interstitial mononuclear cells (CD45 and CD68 positive monocyte/macrophage lineage), rather than bronchiolar epithelium on COX-2 expression [high-COX-2-expressing monocytes, are critically involved in VEGF secretion during VILI. Due to pulmonary epithelial and endothelial cells being evidenced to express COX-2 after various stimulations on resulting lung injury [Here, we focused on the COX-2-expressing Ly6Cing VILI . In our pression . Accordig injury ,34,35,36COX-2 acts as a key role in arachidonic acid metabolism in both physiological and pathological conditions . It is cSix to eight-week-old, C57BL/6 male mice (20–25 g) were purchased from the National Laboratory Animal Center . All of the animal experiments were conducted according to the National Institutes of Health guidelines  that Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital, Chiayi, Taiwan approved  and 2017030202 (15 May 2017)).high monocytes in VILI. Celecoxib  was intraperitoneally administered 1 h prior to mechanical ventilation. Following this, lipopolysaccharide  (20 ng per mouse) was intravenously administered, immediately prior to mechanical ventilation. After tracheostomy, a 20-gauge angiocatheter was introduced into the tracheostomy orifice under general anesthesia by intraperitoneal injection with Zoletil 50 . Anesthesia was maintained with an intraperitoneal injection of Zoletil 50 (10 mg/kg/h) during mechanical ventilation. The mice were placed in a supine position on a heated blanket and then attached to a specialized rodent ventilator  that was ventilated with HTV  for 6 h while the mice were breathing room air with zero end-expiratory pressure. At the end of experiment, the mice were sacrificed via an overdose of anesthetic. The non-ventilated mice served as control. Terminal blood samples of mice were obtained by cardiac puncture and were used for blood gas analysis to assess pulmonary oxygenation and the severity of acute lung injury [We used our previously established murine model of VILI  for studg injury .g for 10 min and the supernatant was collected for a measurement of total protein and VEGF levels. Furthermore, the total number of leukocytes in the sediment was calculated while using a hemocytometer . A 100 μL aliquot of BALF sediment was further smeared on a glass slide for staining with Liu’s stain, which can differentiate and quantify the leukocyte subsets [At the end of the experiment, the catheter that had been inserted into the trachea was lavaged three times with 1 mL of phosphate buffered saline to collect BALF. BALF was centrifuged at 300 ×  subsets . The VEGhigh monocytes were sorted using the FACSAria Fusion cell sorter and analyzed with FACSDiva software version 8.0 (BD Biosciences).As described previously , the mouhigh monocytes that were sorted from the HTV-injured lungs were seeded (1 × 105 cells per well) in the wells of a 96-well plate and cultured in an RPMI medium containing 10% fetal bovine serum in the presence or absence of celecoxib (10 μM). After 1 and 6 h of culture, Ly6Chigh monocyte-conditioned medium was separately collected for measuring the secretion of VEGF by Ly6Chigh monocytes while using an enzyme-linked immunosorbent assay (R&D Systems).Ly6Ct-test. Comparisons among multiple groups were made by one-way analysis of variance with Bonferroni-corrected pairwise post hoc comparisons. All of the statistical analyses were performed with GraphPad Prism software version 5.0 . A p-value of <0.05 was considered to be statistically significant.All the data are expressed as the mean plus or minus the standard deviation (SD). Statistical significance was examined using unpaired Student’s high monocytes, which increase the pulmonary-vasculature permeability and contribute to the pathogenesis of a clinically relevant murine model of VILI with HTV-mechanical ventilation. Therefore, the adjuvant pharmacologic application of COX-2 inhibitors to target the recruitment of COX-2- and VEGF-expressing Ly6Chigh monocytes might provide novel therapeutic insights that will facilitate the treatment and/or prevention of clinical VILI.The present study defines the time course and mechanistic significance of a COX-2-dependent mechanism for the recruitment of activated, VEGF-secreting Ly6C


Intra-orbital hydatid cyst is a very rare pathological entity that affects children and the young adults; it is secondary to the development in the orbit of the echinococcus granulosis tapeworm. Its frequency does not exceed 1% of all cases of hydatid disease. Clinical presentation of intra-orbital hydatid cyst is dominated by proptosis and a decrease in visual acuity, complete surgical excision is difficult, evolution is generally better when the treatment is early before the installation of irreversible optic atrophy. We report one case of a 3 years old girl operated for right intra-orbital hydatid cyst who presented with proptosis and blindness. Complete removal was difficult and puncture of the cyst was performed followed by excision of its membrane with good post-operative results. We also discuss the different epidemiological, clinical, radiological and therapeutic aspects of intra-orbital hydatid cyst and a review of literature of this rare pathology. Echinococcus granulosus tapeworm, whose definitive host are dogs and the intermediate host are sheep. In the parasitic cycle, man is only an accidental intermediate host. Morocco is an endemic country for hydatid cyst where hydatidosis is still prevalent and constitutes a non-exceptional cause of proptosis which represents the major clinical sign associated with a decrease in visual acuity or even monocular blindness and constitutes an emergency surgical treatment because, evolution without treatment will definitely alter the vision [Intra-orbital hydatid cyst is rare, most commonly affecting children and young adults living in rural areas , 2. It ie vision . ThroughA 3 years old girl born from non-consanguineous parents, without any neonatal suffering; with good psychomotor development, from a popular district in one of our cities, without any obvious contact with dogs; presented with exophthalmia associated with unilateral blindness evolving rapidly within 3 months. Clinical examination showed a non-pulsatile, painless, axial, irreducible exophthalmia with no sign of conjunctivitis or keratitis, and right monocular blindness, right ptosis; and fundal examination had objectified right papillary oedema . The resIntra-orbital localization of hydatid cyst is very rare; accounting for less than 0.8 to 1% of all hydatidosis localizations , our casRadiological exploration is mainly based on magnetic resonance imaging (MRI) and computed tomography (CT), but also the ocular ultrasound can be requested, which can reveal a lesion in the form of a fluid mass , homogeneous with a thin wall, clear and regular limits, but sometimes the lesion can be heterogeneous if contains sediments or if infected . With CTIntra-orbital localization of hydatid cyst is very rare, clinical presentation is non-specific, radiological exploration is important and helps to make diagnosis; mainly MRI, and orbital CT scan, the treatment is exclusively surgical avoiding rupture of the cyst and it has a better prognosis with adequate management before the installation of visual field abnormalities and blindness. The diagnosis of orbital hydatid cyst should be considered in the differential diagnosis of unilateral proptosis, especially in patients who live in endemic countries.The authors declare no competing interests.