NPHP1) result in abnormal structure and function of the primary cilia. These deletions can result in a tubulointerstitial kidney disease known as nephronophthisis and retinal (Senior–Løken syndrome) and neurological (Joubert syndrome) diseases. Nephronophthisis is a common cause of end-stage kidney disease (ESKD) in children and up to 1% of adult onset ESKD. Single nucleotide variants (SNVs) and small insertions and deletions (Indels) have been less well characterised. We used a gene pathogenicity scoring system (GenePy) and a genotype-to-phenotype approach on individuals recruited to the UK Genomics England (GEL) 100,000 Genomes Project (100kGP) . This approach identified all participants with NPHP1-related diseases reported by NHS Genomics Medical Centres and an additional eight participants. Extreme NPHP1 gene scores, often underpinned by clear recessive inheritance, were observed in patients from diverse recruitment categories, including cancer, suggesting the possibility of a more widespread disease than previously appreciated. In total, ten participants had homozygous CNV deletions with eight homozygous or compound heterozygous with SNVs. Our data also reveals strong in-silico evidence that approximately 44% of NPHP1 related disease may be due to SNVs with AlphaFold structural modelling evidence for a significant impact on protein structure. This study suggests historical under-reporting of SNVS in NPHP1 related diseases compared with CNVs.Autosomal recessive whole gene deletions of nephrocystin-1 ( NPHP1) was first localised to chromosome 2q13 before a sizeable homozygous deletion was found in this same region1. Saunier et al. and Hildebrandt et al. identified NPHP1 within the minimum deletion interval along with another gene, MALL3. They both noted additional single nucleotide variants (SNVs) in NPHP1 but not in MALL in patients with a heterozygous deletion. Saunier et al. later demonstrated that the NPHP1 gene was flanked by two inverted 358 kilobase (kb) low copy repeats (LCRs) and that these both included a smaller LCR of 45 kb, containing the patient's deletion breakpoints4.Nephrocystin-1  mediated cross-over can occur6. This unequal cross-over results in structural variation, including copy number variants (CNVs) such as duplications, deletions or inversions5.Human chromosome two has arisen from the fusion of two ancestral great ape chromosomes, and the telomere-telomere fusion point is located at 2q13. Telomeres have several repeat sequences, which may explain the presence of these LCRsNPHP1 is localised to the connecting cilia in photoreceptor cells and to the base of the primary renal cilium (known as the transition zone) of renal epithelial cells of the collecting ducts8. Primary cilia are near-ubiquitous sensory organelles that protrude from the cell surface9. Diseases associated with NPHP1 are therefore known as ciliopathies10.NPHP1 is the commonest cause of nephronophthisis (20–25% of cases)13. Nephronophthisis, which means 'vanishing nephron' in Greek, is a tubulointerstitial kidney disease classically divided into three categories, based on the age of presentation: infantile, juvenile, and, less commonly, adolescent14. Ultrasound examination reveals small to normal-sized kidneys, with increased echogenicity due to fibrosis and loss of corticomedullary differentiation. Small corticomedullary cysts may develop later in the disease course due to loss of normal tissue14. The infantile form differs most significantly from the other forms with in-utero onset, oligohydramnios and ESKD in the first two years of life but with enlarged, rather than normal or shrunken kidneys and more widespread cyst development15. Juvenile and adolescent nephronophthisis may be associated with polyuria and polydipsia preceding the development of chronic kidney disease as well as growth retardation14.Homozygous deletion of 16. The retinal phenotype may be mild17. Joubert syndrome (JS) is characterised by cerebellar ataxia, mental retardation, hypotonia, and neonatal respiratory dysregulation18. The cerebellar malformation can be seen on MRI scans as the so-called 'molar tooth sign' (MTS). The malformations are less severe in patients with NPHP1 mutations with milder JS than with other gene causes20.In addition to nephronophthisis, 15% of patients have retinal dystrophy (Senior–Løken syndrome) or neurological (Joubert syndrome) phenotypesNPHP1 is fully penetrant and, therefore, pathognomonic of the nephronophthisis phenotype13. It is well established that nephronophthisis is a leading cause of ESKD in children14. However, more recent data from Snoek et al. identified homozygous deletions in NPHP1 in 0.9% of adult onset ESKD21. One individual first reached ESKD as late as 61 years old. 69% of patients had a prior diagnosis other than nephronophthisis, tubulointerstitial or cystic kidney disease21. Although reported in association with nephronophthisis, small insertions and deletions (indels) and single nucleotide variants (SNVs) are less well characterised in NPHP122.The homozygous deletion of 23. We, therefore, applied our gene-level scoring system, GenePy24. GenePy is a software that transforms variant level data into gene-level data. GenePy generates a score of whole gene pathogenicity for each person by incorporating in silico deleteriousness metrics, population allele frequency and observed zygosity for each variant24. The effect of multiple variants within a gene is combined into a single cumulative gene pathogenicity score for each individual. GenePy scores are continuous but do not follow a normal distribution. Higher GenePy scores are intuitive at a base level as higher scores represent greater pathogenic mutational burden due to rare and deleterious mutations. GenePy scores can be used as the basis to prioritise large numbers of candidate variants.While single variant association testing has successfully identified common disease variants, it remains underpowered for rare variants even with high case numbers25.The UK Genomics England (GEL) 100,000 Genomes Project (100kGP), which completed recruitment in 2018, aimed to sequence the genomes of patients with cancer and rare disease and linked this data with digital clinical records. The aims were to improve clinical diagnosis, tailor therapies, enable new scientific discoveries and link research with a clinically integrated genomic medicine service in the UK National Health Service (NHS)NPHP1 and associated phenotypes using a genotype-to-phenotype approach. We generated GenePy scores for NPHP1 for all individuals recruited to the 100kGP and further incorporated copy number variation (CNV) data. We also interrogated the frequency of CNVs and SNVs in individuals recruited to 100kGP and assessed the evidence for SNVs contributing to the phenotype. We applied a gene first approach or genotype-to-phenotype approach by considering the genotypes of all individuals recruited to the 100kGP irrespective of their disease. We then undertook reverse phenotyping to relate the phenotypic spectrum associated with differing mutations.Using whole-genome sequencing and longitudinal clinical data from the 100kGP project; we aimed to identify pathogenic variation in The 100,000 genomes project was approved by the National Research Ethics Service Research Ethics Committee for East of England—Cambridge South Research Ethics Committee. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects and/or their legal guardian(s) as part of the original study.26. Additional longitudinal phenotype data included the hospital episode statistics (HES) database containing details of all admission, emergency and outpatient appointments at NHS hospitals in England recorded as ICD-10 codes27. The majority of participants provided blood samples, although a small number provided saliva for germline DNA extraction. In addition to germline DNA, some participants recruited to the cancer arm also provided somatic tumour cell DNA.Access to the GEL dataset for the project (research registry ID 109) was approved by the renal GEL clinical interpretation partnership (GeCIP), and all analyses were conducted within the GEL research environment. Patients with GEL-defined eligibility criteria for either rare disease or cancer consented to germline whole-genome sequencing linked to clinical phenotype data, including longitudinal electronic health records. Specific Human Phenotype Ontology (HPO) data were collected at the time of recruitment depending on the participant's recruitment category28. Human Phenotype Ontology (HPO) terms were provided by recruiting Genomic Medicine Centres (GMC) according to the GEL Protocol29.Phenotype data was extracted from LabKey Main programme interim data release v11.030. Samples were aligned to the NCBI Genome Reference Consortium Human Build 38 assembly with decoy sequences. Single sample gVCFs were aggregated using Illumina gVCF genotyper (v.2019.02.26).Genomic data was sourced from an aggregate multi-sample variant call format  main release version 11 (17/12/20). All samples were sequenced with 150 bp paired-end reads in a single lane of an Illumina HiSeq X instrument. Raw sequencing output in the form of a binary base call (BCL) file was processed on the Illumina North Star Version 4 Whole Genome Sequencing Workflow , which comprises the iSAAC Aligner (v. 03.16.02.19) and Starling Small Variant Caller (v.2.4.7). CNVs were called by the Genomics England pipeline using Illumina canvas softwareNPHP1 gene locus ) was selected using bcftools 1.632.A multisource gVCF was extracted from the aggregated gVCF from which the 33.Variants meeting minimum quality standards  were identified using vcftools 0.1.16 and retained for downstream analysis 34. The GenePy score (S) for a given gene (g) and individual (h) is the sum of the effect of all variants (k) where each biallelic variant locus (i) in a gene is weighted according to its predicted deleteriousness (using CADD(Di), zygosity and global allele frequency (gnomAD v3.0)) (f).In order to generate whole gene pathogenicity scores, we applied the GenePy algorithm v.1.3 for both coding and non-coding variants35. Combined Annotation Dependent Depletion (CADD) v1.6 was used to annotate for deleteriousness (D)36. We limited variants to those with CADD PHRED scores greater than 15 (n = 214). This cut-off was chosen as it represents the median value for all possible canonical splice site changes and non-synonymous variants in CADD.The VCF was then annotated using ANNOVAR 1.0 for gnomAD_version 3.0 allele frequency data (f) and the RefGene database of genesNPHP1 was integrated with the whole gene score by arbitrarily assigning (D) as the maximal CADD 1.6 score observed for a stop-gain variant within the NPHP1 gene and allele frequency using gnomAD structural variant v2 (f = 1.798 × 10–3).The GenePy algorithm is streamlined to include indels and SNVs represented in VCF files but does not systematically incorporate CNV data. Therefore, a GenePy score for CNV whole gene deletions of 41. Mutations were identified to be pathogenic following the criteria: (1) the substitution replaces a buried charged residue with an uncharged residue, (2) substitution disrupts all side-chain/side-chain H-bond(s) and/or side-chain/main-chain bond(s) H-bonds, (3) substitution breaks all side-chain/side-chain H-bond(s) and/or side-chain/main-chain H-bond(s) formed by the wild type which was buried, (4) substitution leads to an expansion or contraction of the cavity volume of ≥ 70 Å3, (5) the substitution breaks a salt bridge formed by wild type which was buried. The maximum N–O bond length is 5.0 Å.The mutations identified were modelled both in the Missense 3D Database and using Maestro Suite v13.1 and BioLuminate 4.6 Release 2022-1 NPHP1 GenePy scores identified renal, retinal, or neurological phenotypes amongst the top-ranked individuals  were confirmed to segregate with one variant in each parent. No variants were demonstrated to be de novo. No variants were predicted to affect splicing, including many intronic variants with moderate to high CADD scores. A list of these variants, including heterozygous pathogenic variants and those that failed to segregate or were predicted benign in ClinVar, are included in All SNVs and indels had allele frequencies less than or equal to 4 × 10CNV analysis revealed 424/78,050 participants from 344 families (0.54%) with CNV deletions identified across cancer and rare disease cohorts. The allele frequency of CNV deletions was similar in cancer and rare disease cohorts and twice the rate described in gnomAD SV v2.1  or CKD stage 4 (n = 2). Age at first recorded renal phenotype using either HES ICD-10 data or recruitment HPO terms ranged from 6 to 52 years. Four participants had retinal dystrophy with age at first recorded retinal phenotype ranging from 25 to 54. One participant had no phenotype data available but had been recruited due to 'unexplained renal failure in the young', which required onset of ESKD before the age of 50. One participant was recruited into the cancer cohort for malignant melanoma and had ESKD with tubulointerstitial nephritis and hypertensive renal disease by age 52 and hereditary retinal dystrophy by age 54. All ten patients with CNV deletions were deemed pathogenic by ACMG guideline standards due to the predicted null variant nature of the gene deletion giving very strong evidence of pathogenicity.Eight participants had a phenotype consistent with renal or retinal disease and homozygous or compound heterozygous SNVs. These included a homozygous stop-gain mutation (two siblings from one family), heterozygous CNV deletions with a missense mutation (two siblings from one family), two different homozygous missense mutations , and two different possible compound heterozygous missense mutations . The participants with homozygous stop gained and homozygous missense mutations and three of the ten with homozygous CNV deletions were known to have consanguinity. Of these eight the two siblings with homozygous stop-gained variants could be reported as pathogenic according to ACMG guidelines due to the null variant. The other SNVs were rare (4.00E−04) or novel missense variants with strong in-silico evidence of pathogenicity with CADD scores of between 18 and 28.9.NPHP1-related disease but without a documented phenotype. These included six with possible compound heterozygous missense mutations and two compound heterozygous CNV deletions in-trans with a missense mutation. One participant without a documented renal or retinal phenotype was recruited to the cancer cohort. The other was the relative of a participant recruited with intellectual disability and no phenotype data available for renal or retinal disease.An additional eight participants from eight families had recessive genotypes consistent with monogenic Many heterozygous CNV deletions (n = 414) were discovered, including six in the top 50 GenePy rankings. Two heterozygous CNV deletions were identified in participants recruited to the cancer cohort, but no renal or retinal phenotype was recorded. However, the deletions were in-trans with an intronic variant with a CADD 1.6 PHRED score of 15.56 and found in an active enhancer mark (H3K27ac). The remaining heterozygous CNVS were joint ranked 593 and not found with any additional predicted pathogenic variants by CADD 1.6.44. In short, the AlphaFold algorithm is an AI system based on the analysis of contact maps of co-evolving residues extracted from multiple sequence alignments (MSAs) of sequences, which feed two neural networks: one trained on Protein Data Bank (PDB) structures to predict interatomic angles and distances; another, trained to score the geometry and structural accuracy. This model identifies five different structural regions within the NPHP1 polypeptide chain NPHP1-related disease and renal or retinal phenotypes. Of these, ten participants with homozygous NPHP1 deletions could be considered pathogenic according to ACMG guidelines. Eight patients had homozygous and compound heterozygous SNVs, including two heterozygous variants of uncertain significance (VUS) in-trans with CNV deletions. Of these, only the two with homozygous stop gain mutations can be classified as pathogenic according to ACMG criteria.Eighteen participants were identified with genotypes consistent with a diagnosis of recessive 46. AlphaFold structural modelling of these SNVs additionally predicted significant impact on protein structure.The other SNVs were rare (4.00E−04) or novel missense variants with strong in-silico evidence of pathogenicity. The CADD PHRED scores were between 18 and 28.9. At the risk of losing some causative variants, we chose a cut off CADD score of 15 and above. This cut-off was chosen as it represents the median value for all possible canonical splice site changes and non-synonymous variants in CADD. Variants with CADD PHRED scores over 15 are predicted to be in the top 0.5% of most deleterious substitutions that can occur in the human genome. However, as is true of missense variants in many other genes they cannot be officially classified as clinically pathogenic without expensive and time consuming in-vitro or in vivo functional assays. This poses a wider problem with the bottle neck to clinical interpretation now moving from high through put genomics to a lack of high throughput functional assays to confirm pathogenicity. Given these variants are rare or even private to a specific family, the cost of developing 'well-established in vitro or in vivo functional studies' is likely to be prohibitive and result in a loss of translational benefit to patients and their families. Where functional experimental evidence is available it supports roles of some protein domains in apoptosis and cell adhesionNPHP1-related disease but no documented renal or retinal phenotype. One was recruited with cancer, and three were 'unaffected' relatives. Generally, phenotype data are scarce for participants recruited to cancer and for rare disease participants other than the proband. The GEL recruitment process for cancer participants does not routinely require documentation of kidney function, even though this would invariably have been tested in all patients undergoing treatment for their cancer. 'Unaffected' relatives with no phenotype recorded at recruitment and no longitudinal ICD-10 HES data could be due to either not being clinically evaluated, having subclinical disease, or having a clinical disease that is not documented, that may present later or is not fully penetrant.An additional eight participants had a recessive SNV genotype consistent with monogenic NPHP1 CNV deletions from Snoek et al. suggests that individuals may not reach end-stage kidney disease until the seventh decade21. Longitudinal follow up of these participants using HES data from GEL will be required to clarify penetrance and confirm other cases.When assessing compound heterozygosity, the difficulty is that some variants may lie on the same chromosome. This cannot currently be assessed with short-read data such as that used by Genomics England. We confirmed the presence in-trans in one participant through parental assessment. Their young age may explain the lack of phenotype data for probands with compound heterozygous variants. They may yet develop significant disease in their lifetime (two were under 15 years old). Array-based data on homozygous NPHP1 and related diseases. We therefore applied a more objective, agnostic genotype-to-phenotype approach. A significant benefit over a case–control approach is that it precludes the need to exclude participants from analyses based on predicted ancestry or relatedness. This is particularly important given the under-representation of individuals from non-European ancestries in the genetic literature. As with all approaches that implement short read data, this approach is subject to the same limitations. This includes reduced sensitivity to detect variants in highly variable regions and absence of phase representation. A lack of phase data is particularly problematic in adult-onset disease where parent DNA may not be available to determine zygosity, therefore limiting the ability to assess compound heterozygous variants. As with all large-scale data, GenePy scoring is dependent upon data quality and the elimination of systematic bias and technical artefacts. However, the genomics England data used was of high integrity, with all samples sequenced with 150 bp paired-end reads in a single lane of an Illumina HiSeq X.The large number of participants in the 100kG project offered a unique opportunity to identify the full spectrum of pathogenic variation in PDE6B) other than NPHP1. This highlights the possibility that some participants have more than one monogenic molecular diagnosis. In one whole exome study of 7374 patients, 101 (4.9%) had diagnoses that involved more than one locus, and associated phenotypes could be distinct or overlapping47. All patients with more than one genetic diagnosis were known to be consanguineous.Our data support the benefit of a gene first approach that allows the identification of genotypes and associated phenotypes unbiased with respect to their phenotype. This approach can be applied to any gene and includes participants who may yet to develop disease. Notably, the genotype–phenotype approach did not miss any individuals reported as solved by the GEL clinical interpretation pipeline at the time of writing. According to GMC exit questionnaire data, nine participants from seven families were reported by GEL using the clinical interpretation pipeline. However, one was reported for another retinal gene  may yet demonstrate these phenotypes.Homozygous CNV deletions of NPHP1) in the UK 100,000 genomes project irrespective of relatedness or ancestry. Supportive data from AlphaFold structural modelling suggests that up to 44% of diagnoses of NPHP1-related disease may be caused by SNVs in the addition to the well described CNV deletions. Increasing availability of phased genomic data from long read sequencing is expected to further the power of this approach and allow confident assessment of compound heterozygous variants. Longitudinal follow up will provide data on the ability to predict future disease states in high GenePy scoring individuals.The gene first (genotype-to-phenotype) approach combined with a gene pathogenicity score (GenePy) enabled the identification of the full molecular genetic spectrum of nephrocystin-1 (Supplementary Information.


SlATG8 family members during tomato fruit ripening. Transmission electron microscopy observations and dansylcadaverine (MDC) staining confirmed the presence of autophagy at the cellular level in tomato fruits. Furthermore, the overexpression of SlATG8f induced the formation of autophagosomes, increased autophagic flux within tomato fruits, and effectively enhanced the expression of ATG8 proteins during the color-transition phase of fruit ripening, thus promoting tomato fruit maturation. SlATG8f overexpression also led to the accumulation of vitamin C (VC) and soluble solids while reducing acidity in the fruit. Collectively, our findings highlight the pivotal role of SlATG8f in enhancing tomato fruit ripening, providing insights into the mechanistic involvement of autophagy in this process. This research contributes to a better understanding of the key factors that regulate tomato fruit quality and offers a theoretical basis for tomato variety improvement.Autophagy is a highly conserved self-degradation process that involves the degradation and recycling of cellular components and organelles. Although the involvement of autophagy in metabolic changes during fruit ripening has been preliminarily demonstrated, the variations in autophagic flux and specific functional roles in tomato fruit ripening remain to be elucidated. In this study, we analyzed the variations in autophagic flux during tomato fruit ripening. The results revealed differential expression of the  Autophagy is a highly conserved intracellular self-degradation system in eukaryotic organisms that is utilized to eliminate and recycle cellular components, thereby breaking down toxic substances or damaged organelles and reclaiming essential nutrients ,2. CurreATGs). Initially discovered in yeast [ATGs have been subsequently identified in animals and plants. The proteins encoded by ATGs participate in various stages of the autophagy process [ATG8 function prevents autophagosome formation in yeast and other fungi [ATG8 gene redundancy. On the other hand, the overexpression of ATG8 promotes autophagosome formation in Arabidopsis and rice [The formation of autophagosomes is governed by a plethora of autophagy-related genes  components, ATG4 activity, autophagy receptor NBR1, protease Lon1, and protease Lon2 gene expression and protein content changes in two pepper varieties. Their findings confirmed the presence of autophagic structures in pepper fruit, highlighting their crucial role in fruit maturation and quality formation [Research has indicated the significant role of autophagy in fruit ripening . Transcrturation , implyinormation . Similarormation . These sSolanum lycopersicum L.), one of the most esteemed vegetables globally, is renowned for its diverse flavors, forms, colors, and abundant nutritional value [ATG8 family during tomato fruit ripening, we demonstrate the existence of autophagic bodies within tomato fruit at the cellular level. Additionally, we observe the changes in ATG8 protein content necessary for autophagosome formation. Overexpression of SlATG8f leads to increased autophagic flux in tomato fruit, accelerating fruit ripening, upregulating ethylene-related genes, and enhancing fruit quality. Collectively, these findings suggest that autophagy is involved in regulating tomato fruit ripening and senescence, thereby enriching our understanding of the autophagic mechanisms in tomato fruit ripening and providing insights into the key factors that regulate fruit quality, ultimately contributing to the improvement of tomato varieties.Tomato  was employed to quantify the transcript levels of SlATG8 family members during different stages of Micro-Tom tomato fruit maturation, including the green ripe, breaker, orange ripe, and red ripe stages  under the control of the 35 S promoter. Quantitative real-time polymerase chain reaction (qRT-PCR) results demonstrated a substantial increase in SlATG8f gene expression in the SlATG8f-overexpressing (OE) plants, reaching up to 16-fold higher levels compared to the wild-type (WT) plants  plants A.SlATG8f OE plants and WT fruits, a notable dissimilarity was observed upon slicing the fruits. The pericarp parenchyma of the SlATG8f OE plants’ fruits exhibited accelerated development compared to that of the WT fruits with the pericarp parenchyma being more plump and filled in the SlATG8f OE plants’ fruits than in the WT fruits. In the early stages of fruit development, the pericarp parenchyma in the WT fruits was relatively scant, whereas the pericarp tissue of the SlATG8f OE plants’ fruits was already completely filled. By the 35th day of fruit development, both the SlATG8f OE plants and WT fruits had entered the liquefaction phase. However, the SlATG8f OE plants’ fruits displayed a higher degree of liquefaction in their pericarp parenchyma. In the late stages of fruit development, both the SlATG8f OE plants’ and WT fruits were fully matured, and their pericarp parenchyma had undergone significant liquefaction with no conspicuous differences between them. Nonetheless, the pericarp parenchyma tissue of the SlATG8f OE plants’ fruits remained fuller compared to that of the WT fruits.Phenotypic changes in fruit development were observed among the different tomato plants during fruit ripening B. While SlATG8f enhances autophagic activity in tomato fruit, this study employed MDC staining and protein immunoblotting (Western blotting) to assess the changes in autophagic activity between the SlATG8f OE plants’ and WT fruits at different stages of ripening , soluble solids content (SSC), titratable acidity (TA), and tomato lycopene levels. The results revealed notable variations in these components. At all ripening stages of the SlATG8f OE plants’ fruits, the content of both ascorbic acid and soluble solids was higher compared to that of the WT fruits , breaker (day 35), orange ripe (day 40), and red ripe (day 45). For each stage, 4–5 fruits were sampled, and the middle portion of the peel and flesh was harvested, frozen in liquid nitrogen, and stored at −80 °C. Gr: green ripe; Br: breaker; Or: orange ripe; Re: red ripe.The full-length SlATG8f coding sequence (CDS) was amplified from tomato cDNA using the specific primer  into pBWQuantitative real-time PCR was employed to study the expression of SlATG8s and ethylene-related genes in tomato fruits. Frozen tomato fruit samples were used for RNA extraction with a TIANGEN kit , and cDNA synthesis was performed using a reverse transcription kit . Specific primers were designed using Primer 5.0 software with the Actin gene selected as the internal reference . qRT-PCR3 pieces, fixed in electron microscopy fixative solution  at 4 °C for 2–4 h, and then dehydrated using a series of acetone gradients. The dehydrated tissues were infiltrated and embedded in mixtures of dehydrating agents and epoxy resin in ratios of 3:1, 1:1, and 1:3. Ultrathin sections (50 nm) were obtained using an ultrathin slicer, and these sections were placed on copper grids. The sections were stained with uranyl acetate for 10–15 min and then with lead citrate for 1–2 min at room temperature. Transmission electron microscopy was performed using a JEM-1400PLUS  instrument.Tomato fruits were soaked in a 100 nmol/L rapamycin solution for 12 h to induce autophagy. The fruit tissues were rapidly cut into 1–2 mmTomato fruits were soaked in a 100 nmol/L rapamycin solution for 12 h to induce autophagy. The tomato fruit sections were prepared according to a previous protocol . The secTomato fruits were soaked in a 100 nmol/L rapamycin solution for 12 h to induce autophagy. The fruit tissues were frozen in liquid nitrogen and used for total protein extraction using a kit . SDS-PAGE gel electrophoresis was performed  to separate the proteins. The membrane was blocked with 5% skim milk for 2 h and then washed with TBST. The PVDF membrane was cut into strips and incubated with primary antibodies (1:1000) overnight at 4 °C. After washing with TBST three times, the membrane was incubated with secondary antibodies (1:1000) against rabbit ATG8a polyclonal antibody  for 1 h at room temperature. Chemiluminescence was detected using a chemiluminescence detection kit .v/v) and shaken. After adding 15 mL of water and allowing for phase separation, the organic phase (hexane) absorbance was measured at 503 nm to calculate the lycopene concentration. The molar extinction coefficient was 17.2 L mol−1·m−1, and the units were expressed as mg·kg−1 based on the fresh weight [The lycopene content of the tomato fruits was determined according to Sun et al.’s method. Five grams of tomato flesh and peel tissue were frozen and ground into powder. The powder was mixed with 50 mL of hexane–acetone–ethanol , and the liquefaction rate of the locular gel tissue was faster, indicating that the SlATG8f gene positively regulates tomato fruit development. The MDC staining and electron microscopy observations revealed that the number of autophagosomes in the SlATG8f OE plants’ fruits was significantly higher than that in the WT, especially during the breaker stage, with a substantial increase in the number of autophagosomes and autolysosomes in the fruit flesh cells, indicating enhanced autophagic activity. Western blot results demonstrated a significant increase in ATG8 protein expression during the breaker stage in the SlATG8f OE plants’ fruits, and the expression level of SlATG8f correlated positively with autophagic activity. Overexpression of SlATG8f induced the formation of autophagosomes in the tomato fruit cells, enhancing autophagic activity. The increased autophagic flux due to SlATG8f overexpression accelerated fruit ripening and upregulated the expression of ethylene-related genes, regulating fruit ripening. Taken together, our findings suggest that autophagy is involved in regulating tomato fruit ripening and senescence. These discoveries contribute to a deeper understanding of the role of autophagy in the process of tomato fruit ripening, elucidate the key factors that regulate fruit quality, and provide a theoretical basis for improving tomato varieties.In this study, quantitative analysis of the expression of the


Cystic ovarian disease is an important ovarian disorder that leads to anovulatory infertility in dairy cows. In the present study, we used spontaneous ovarian follicular cysts to show the first evidence that the deregulation of Hippo pathway effector yes-associated protein expression and/or activity can be a potential key to better understand cystic ovarian disease pathogenesis. This finding also allows us to point towards Hippo pathway as a potential therapeutic target for the treatment of this major ovarian disorder in cattle.CTGF, BIRC5 and ANKRD1 in GCs from follicle cysts in comparison to non-cystic large follicles. Taken together, these results provide considerable insight of a completely novel signaling pathway that seems to play an important role in ovarian cystic disease pathogenesis in dairy cattle.Cystic ovarian disease (COD) in dairy cattle is characterized by preovulatory follicles that become cysts, fail to ovulate and persist in the ovary; consequently, interfering with normal ovarian cyclicity. The intraovarian key players that orchestrate the alterations occurring in the preovulatory follicle and that culminate with cyst formation and persistence, however, remain uncertain. Interestingly, the Hippo pathway effector yes-associated protein (YAP) has been described in humans and mice as a key player of anovulatory cystic disorders. To start elucidating if YAP deregulation in ovarian follicle cells can be also involved in the pathogenesis of COD, we have generated a series of novel results using spontaneously occurring cystic follicles in cattle. We found that mRNA and protein levels of YAP are significantly higher in granulosa (GCs) and theca cells (TCs) isolated from cystic follicles (follicular structures of at least 20 mm in diameter) in comparison to respective cell types isolated from non-cystic large follicles (≥12 mm). In addition, immunohistochemistry and Western blot analyses used to determine YAP phosphorylation pattern suggest that YAP transcriptional activity is augmented is cystic GCs. These results were confirmed by a significant increase in the mRNA levels encoding for the classic YAP-TEAD transcriptional target genes  The fertility of high-yielding dairy cows has been declining for decades due to many factors, and notably ovarian malfunctions are at the top of the list ,2. AmongOvarian cysts normally develop from preovulatory follicles that have failed to ovulate, they persist in the ovary and thus interfere with normal ovarian cyclicity causing anovulatory infertility . AlthougThe Hippo intracellular signaling pathway is evolutionarily highly conserved with well-known roles in cell differentiation, proliferation and apoptosis in several tissues, particularly during embryogenesis ,9. The cA number of studies indicate the importance of Hippo effectors to ovarian physiology in adult animals, including cattle ,15,16. IThe reagents used in the present study were obtained from Thermo Fisher Scientific  except where otherwise stated.Ovarian follicle cysts were selected based on macroscopic evaluation, and were identified as follicles larger than 20 mm and with a follicle wall of up to 3 mm thickness ,20. Cystg for 10 min each. After assessing GC viability (with 0.4% Trypan blue stain) and counting the number of viable cells, 1 × 106 viable cells were placed in RLT buffer for RNA extraction or in M-PER® mammalian protein extraction reagent for protein extraction, and stored at −80 °C. Following GCs removal, some follicles were then used for TCs isolation. Briefly, the TCs layer was dissected from the surrounding stroma and washed several times with PBS plus heparin-enriched solution to remove all remaining attached GCs. Then, samples were aliquoted (50 mg each) for total RNA and protein extraction as described above. Bovine ovaries were collected at different days from random adult cows at a local abattoir and were transported to the laboratory in PBS at 35 °C containing penicillin (100 IU/mL), streptomycin (100 μg/mL) and fungizone (1 μg/mL). For histology, ovaries were fixed in formalin (10% buffered), paraffin-embedded, stained in H&E and evaluated histologically. An approximate volume of 400 µL of follicular fluid was collected from each follicle and stored at −80 °C for hormone assay. GCs were then collected from the cystic and non-cystic follicles by aspiration and washed twice by centrifugation at 219× For immunohistochemistry (IHC) analyses, bovine ovaries and sample selection for experimental or control groups were performed as described above. At the laboratory, entire ovaries (at least four per group) were fixed in 10% formaldehyde solution for 24 h, rinsed and dehydrated in alcohol until embedded in paraffin. Serial sections were prepared (at a thickness of 3 µm) followed by deparaffinization, rehydration, sodium citrate heat-mediated antigen retrieval, peroxidase block and protein blocking (10% goat for 30 min), and then slides were probed with primary antibody against total and phosphorylated forms of YAP or against Pan-TEAD  overnighH2AFZ, GAPDH and RPL19) with the 2−∆∆Ct method and correction for amplification efficiency [Total RNA from GCs and TCs from control and cystic follicles were extracted using the Total RNA Mini Kit  according to the manufacturer’s protocol. Reverse transcription was performed using 50 ng of total RNA and the SuperScript Vilo cDNA synthesis kit. Real-time qPCR was performed using Advanced supergreen qPCR mastermix  and a CFX96 Touch™ instrument . Each PCR reaction consisted of 7.5 μL of SsoAdvanced SYBR Green PCR Master Mix, 2.3 μL of water, 4 μL of cDNA sample and 0.6 μL (10 pmol) of gene-specific primers . Cyclingficiency . Primers® mammalian protein extraction reagent according to the manufacturer’s instructions and protein levels were quantified using the Pierce™ BCA Protein Assay Kit. Halt™ Protease and Phosphatase Inhibitor Cocktails were added to the samples final solution to avoid protein degradation. Samples (20–40 μg) were resolved on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to Hybond-P PVDF membrane . Membranes were then probed at 4 °C overnight in 5% BSA in TTBS with different primary antibodies , progesterone (P4) and testosterone (T) were measured by liquid chromatography–tandem triple quadrupole mass spectrometry (LC-MS/MS) in the Endocrine Technologies Core (ETC) at the Oregon National Primate Research Center (ONPRC) as previously described . Brieflyt-tests were used to compare the two experimental groups. All data are presented as means ± SEM and variables that were considered statistically significant at p < 0.05 are represented with an asterisk symbol (*).All analyses were performed using four to ten independent follicle samples per group (cystic or non-cystic), each isolated from ovaries collected at different days. The statistical analyses for all assessments were performed using GraphPad Prism software version 9.3.1 . Data from mRNA abundance or target protein levels that were not normally distributed (Shapiro–Wilk test) were transformed to natural logarithms. Two-tailed PGRb), androgen receptor (AR), steroidogenic acute regulatory protein (STAR), luteinizing hormone (LHCGR), follicle stimulating hormone receptor (FSHR) and estrogen receptors type 1 (ESR1) and 2 (ESR2). Abundance of mRNA encoding PGRb, AR and STAR was significantly increased in GCs from cysts compared to control GCs, and mRNA levels for both LHCGR and FSHR were significantly lower in cyst CGs in comparison to controls  proteins in both non-cystic and cystic follicles . ImmunosInterestingly, GCs from cystic follicles seemed to present not only a lower number of nuclei positive for phospho-YAP, but also the intensity of such a signal in the nucleus and in the cytoplasm of these cells was weaker when compared to respective cells from the control group. Most interestingly, while the theca cell layer from cyst follicles presented some extent of positive signal for phospho-YAP (predominantly in their cytoplasm), a poor to absent signal for phospho-YAP was detected in theca cells from non-cystic control follicles.p < 0.05; To complement our IHC analyses and to better assess if YAP total protein levels were indeed augmented in GCs isolated from cyst group samples, we then employed Western blot analyses. The results demonstrated that total YAP protein levels were significantly higher in GCs from cystic follicles in comparison to GCs from control follicles, but also that phospho-YAP abundance was decreased (TEAD 1–4 and the classic YAP-TEAD target genes connective tissue growth factor (CTGF), baculoviral IAP repeat-containing protein 5 (BIRC5), cysteine-rich angiogenic inducer 61 (CYR61) and ankyrin repeat domain 1 (ANKRD1) as a measure of Hippo pathway activity. The results showed that the mRNA abundance for TEAD4 was significantly increased in the cyst group  were not statistically different between the control and cyst group  characterization in cattle; nevertheless, the core players that orchestrate the alterations occurring in the large antral follicle and that culminate with cyst formation and persistence remain uncertain. In the present study, we used spontaneous ovarian follicular cysts to show the first evidence that the deregulation of Hippo effector YAP expression and/or activity can be a potential key to better understand COD pathogenesis. We show herein that YAP expression and transcriptional activity is significantly increased in granulosa cells of cystic follicles in comparison to that observed in non-cystic large preovulatory follicles. Conversely, YAP-TEAD-related transcriptional activity in theca cells from cysts is highly compromised, most likely due to lower expression levels for TEADs observed in these cells.PGRb, AR and STAR was significantly increased in our cyst groups in comparison to controls. In addition, a known characteristics of COD follicles is their low responsiveness to gonadotropins due to decreased gonadotropin receptors in GCs [LHCGR and FSHR were significantly lower in our cyst group samples in comparison to controls. Phenotypically, bovine ovarian cysts are normally defined as anovulatory ovarian structures with a cavity greater than 20 mm in diameter and that persist for at least 10 days in the absence of a functional corpus luteum. The main macroscopic difference between follicular cysts and luteinized follicular cysts is based on their follicle wall thickness which is less than 3 mm in follicular cysts and greater than 3 mm in luteinized follicular cysts ,27,28. Ts in GCs ,31,32,33Ctgf in mouse GCs increases Lhcgr mRNA levels and concomitantly increases GCs apoptosis [Although the experimental samples analyzed herein do not allow us to state conclusively that YAP expression and transcriptional activity has a causal effect on COD pathogenesis, the potential functions exerted by CTGF, BIRC5 and ANKRD1 (YAP-TEAD-target genes that were increased in cystic GCs herein) may represent strong evidence of its potential key role in cyst formation and persistence. In addition to the lower expression of gonadotropin receptors described previously, GCs in bovine ovarian cysts generally have low rates of proliferation and of apoptosis ,35. Intepoptosis . In addipoptosis . BIRC5 ppoptosis . Finallypoptosis . Taken tThe main GCs-related results showed herein are compatible with important findings in humans. First, YAP was identified as a susceptibility gene for polycystic ovarian syndrome (PCOS) in humans ,40,41, aCTGF, CYR61 and ANKRD1) decreased in TCs from cystic follicles. One explanation is the decrease in TEAD expression observed in these cells, such that even in the presence of increased YAP the availability of TEAD proteins became a limiting factor and abrogated expression of the Hippo target genes. However, the mechanism behind such a decrease and its relevance for COD pathogenesis remains to be elucidated. A puzzling finding of our study is the paradox between YAP expression vs. YAP-TEAD transcriptional activity in theca cells from cystic follicles. In these cells, total YAP protein abundance increased with no change in phospho-YAP, suggesting an accumulation of non-phosphorylated YAP, and it is this unphosphorylated YAP that can accumulate in the nucleus and activate TEAD-dependent transcription . HoweverWe provide a novel insight that the Hippo pathway effector YAP may play a key role in the pathogenesis of cystic ovarian disease in dairy cattle. Further studies are still required to establish its causal effect in the etiology of COD, and to determine the factors/conditions leading to YAP deregulation in ovarian follicle cells. Nonetheless, this first evidence points to YAP as a potential therapeutic target for the treatment of this major ovarian disorder in cattle.


Pit-1 tumours are derived from neoplastic cells of either somatotroph, lactotroph or thyrotroph cell lineages, but there are also distinct mixed tumours and plurihormonal tumours within this category as described within the 2022 edition of the WHO classification of pituitary tumours. Plurihormonal tumours and thyrotroph adenomas are transcriptionally similar and grouped together to discuss in this review, although it is clear an immature type of plurihormonal tumour exists which are more commonly associated with refractory disease. Management of residual or recurrent disease should follow that of other aggressive pituitary tumours, although a trial of somatostatin analogue therapy is certainly warranted before considering temozolomide therapy. The Pit-1 lineage of pituitary tumours has evolved in the 2022 WHO classification to encompass tumours derived not only from mature somatotroph, lactotroph and thyrotroph cells, but recognising distinct plurihormonal types and tumours originating from precursor cells  and “Mature plurihormonal Pit-1 lineage” (MPL) tumours to address the heterogeneity within the group. IPL -secreting adenomas (thyrotroph adenomas (TA) or “TSHomas”) account for 2–3% of pituitary adenomas, although higher prevalence rates in the past 2 decades are attributed to a rise in detection of microadenomas –8. TAs m were PC , 12. Agg were PC –20. In a1 subtype, 22.There appears to be a male predilection among aggressive TAs, with 2/3 PCs and all 5 across ESE surveys being male, compared with a 1.07 F:M ratio among all published TA cases . This seTranssphenoidal pituitary surgery remains first-line treatment for TAs as described in European Thyroid Association guidelines published in 2013 . OverallPit-1 plurihormonal tumours comprise both mature (MPL) and immature (IPL) types now recognised in the WHO 2022 classification. These tumours have gene expression profiles that are closely aligned with TA, although IPL more closely resembles the previously known silent subtype 3 adenoma and may account for the poorer prognosis often attributed to these Pit-1 lineage tumours. In cases with residual or recurrent disease following surgery, a trial of SSA is warranted and RT may be effective. Low MGMT expression may be seen more frequently in IPL but data on temozolomide efficacy is limited but should be first-line chemotherapy in the absence of other known effective therapies.


Idiopathic pulmonary fibrosis (IPF) is the most prevalent progressive idiopathic interstitial pneumonia. It has a poor prognosis  and typically presents in older individuals (mean age 74 years) [1]. Systemic hypertension is highly prevalent and the leading contributor to all-cause death and disability worldwide. Hypertension is a risk factor for common comorbidities of IPF including ischaemic heart disease, and >50% of individuals with IPF have systemic hypertension (defined as ≥130 mmHg systolic blood pressure (SBP) and/or ≥80 mmHg diastolic blood pressure (DBP) [2]). High blood pressure can lead to organ fibrosis or can be a consequence of artery stiffening due to fibrosis [3], and studies have implicated common pathways in both IPF and blood pressure regulation, such as transforming growth factor (TGF)-β signalling and the renin–angiotensin system [4]. The discovery of shared genetic associations, with sometimes different directions of effect, has implications for drug target discovery for idiopathic pulmonary fibrosis and systemic hypertension https://bit.ly/45IqbRv To the Editor:Idiopathic pulmonary fibrosis (IPF) is the most prevalent progressive idiopathic interstitial pneumonia. It has a poor prognosis  and typically presents in older individuals mean age 74 years) . Systemi4 years .−5 between IPF and either SBP or DBP; this was performed by identifying signals at p<10−5 in both datasets, and then performing colocalisation. We report signals with a posterior probability of >80% for a shared causal variant (coloc H4) as significant. Signals were mapped to a gene either using prior published evidence or using Open Targets Genetics (OTG). OTG utilises distance from gene, expression quantitative trait loci (QTL), splice QTL and functional annotation to suggest the most likely variant-to-gene mapping.We utilised the largest available genome-wide association studies (GWAS) of clinically defined IPF , and SBPMAD1L1, GOLPH3L/HORMAD1 and at 17q21.31) had the same direction of effect on risk of IPF and raised blood pressure (hypertension) and five  had opposite effects, with the allele associated with increased risk of IPF being associated with decreased blood pressure   or DBP . The genome-wide colocalisation analysis identified 18 regions of overlap, of which seven had a posterior probability of a shared causal variant >80% for IPF and blood pressure . In addi allele) .Our study identified shared genetic associations between IPF and blood pressure traits with genetic variants at some loci increasing the risk of both IPF and hypertension, while at other loci, the same genetic variants increased risk of one trait while being protective against the other.MAD1L1 (mitotic arrest deficient 1 like 1) encodes a component of the mitotic spindle assembly checkpoint and is a shared and consistent signal of association between IPF and hypertension. Common and rare genetic variants at the KIF15 (kinesin family member 15) gene [SPDL1 (spindle apparatus coiled-coil protein 1) [15) gene , and a rotein 1)  have alsotein 1) , 17.DEPTOR (DEP domain-containing mTOR-interacting protein) encodes a mammalian target of rapamycin (mTOR) inhibitor and decreased DEPTOR gene expression was associated with increased risk of IPF [k of IPF . Targetik of IPF . Our fin−8 will have partly mitigated this, but it is likely that more shared signals will be discovered as sample sizes increase. We have used published datasets that undertook adjustment for appropriate demographic covariates and restriction to individuals of European ancestry. Differences in age, sex and smoking history between the datasets may mean that we may have missed additional shared signals in our study. For example, where there may be interaction effects for either trait. None of the shared signals we highlight are associated with smoking.There was a significant difference in sample size for the GWAS used in our analyses and this will have affected the statistical power to detect signals in the smaller IPF dataset, meaning that there may be additional shared signals that we have not detected. Our genome-wide approach utilising a more lenient threshold than the commonly used genome-wide threshold of p<5×10We only report signals with a single shared causal variant for IPF and blood pressure. It is plausible that distinct causal variants could exert their effect through the same nearby gene or different genes in the same molecular pathway; however, this study was not designed to detect such effects. It is likely that genetic effects may be tissue-specific and that different regulatory pathways may be involved in the expression of the same gene in different tissues.vice versa), it is important to understand the mechanisms of such shared pathways and to ensure that genetically indicated potential adverse effects are proactively monitored in future clinical trials for new drugs.Our findings provide support that IPF and systemic hypertension have shared mechanisms which may promote both fibrosis and hypertension, as well as others where there are opposite directions of effect. Given the frequency of hypertension and ischaemic heart disease as comorbidities in IPF, and the effect of potential antifibrotics on blood pressure (and


Middle Stone Age (MSA) technologies first appear in the archaeological records of northern, eastern and southern Africa during the Middle Pleistocene epoch. The absence of MSA sites from West Africa limits evaluation of shared behaviours across the continent during the late Middle Pleistocene and the diversity of subsequent regionalized trajectories. Here we present evidence for the late Middle Pleistocene MSA occupation of the West African littoral at Bargny, Senegal, dating to 150 thousand years ago. Palaeoecological evidence suggests that Bargny was a hydrological refugium during the MSA occupation, supporting estuarine conditions during Middle Pleistocene arid phases. The stone tool technology at Bargny presents characteristics widely shared across Africa in the late Middle Pleistocene but which remain uniquely stable in West Africa to the onset of the Holocene. We explore how the persistent habitability of West African environments, including mangroves, contributes to distinctly West African trajectories of behavioural stability. The late Middle Pleistocene site of Bargny, Senegal, documents stone tool trends seen across contemporary sites in Africa but which, in West Africa, remain uniquely stable into the Holocene. Palaeoenvironmental data suggest that persistently stable environments in West Africa through the Late Pleistocene, including estuarine refugia, may have supported consistent behavioural responses. Homo sapiens in the fossil record2. Recent studies have highlighted the substantial influence of population structure within and between regions in the mosaic appearance of Homo sapiens morphology and subsequent patterns of population diversity4. The spatial and temporally disjunctive nature of the transition from preceding Acheulean technologies to the appearance of the MSA supports a comparable scenario for cultural evolution, perhaps best evidenced by the late persistence of Acheulean populations at Mieso (Ethiopia)6 around 60 ka after the appearance of MSA technologies elsewhere in eastern Africa2. The transition to MSA technologies is marked by a change in the dominant focus of lithic-reduction practices from production of larger bifacial tools to prepared core reduction and use of retouched flake tool kits, notable increases in transport of raw materials across the landscape and the appearance of additional categories of artefacts within archaeological assemblages, such as ochre2. Examining the early appearances of the MSA within the Middle Pleistocene is critical to investigate regional trajectories of behavioural change within their palaeoecological context and inter-regional connectivity in both behaviour and environmental adaptation that complement studies of demographic variability.The appearance of the Middle Stone Age (MSA) during the Middle Pleistocene ) marks a major cultural change in African prehistory. The earliest occurrence of MSA technologies around 300 ka is broadly contemporaneous with the appearance of 7), eastern Africa  and southern Africa (Kathu Pan10). Early-MSA occupations in Africa become more numerous in MIS 7 (243–191 ka) and to a lesser extent in MIS 6 , a substantial elaboration of material activity  and the occupation of a broader range of habitats14, including desert27, high altitudes29, tropical forests31 and coastlines34. Establishing the antiquity of the MSA in poorly understood regions such as West Africa is critical to assess both the earliest appearance of similar novel behaviours, to provide a benchmark against which to evaluate subsequent regionalized patterns of cultural innovation and change and to examine how MSA populations engaged with and adapted to distinct ecological settings.The earliest occurrences of MSA sites across Africa appear in Marine Isotope Stage (MIS) 9 (337–300 ka), and the initial stages of MIS 8 (300–243 ka) in northern Africa , indicating occupation of the region from the start of MIS 5 (130–71 ka). The majority of MSA sites in the region date between MIS 4 (71–59 ka) and MIS 2 (28–11 ka) (refs. 41), with the youngest MSA occupation across Africa reported from the onset of the Holocene from Saxomununya, about 11 ka (ref. 37). West African MSA sites are typically found in modern Sahel or savannah habitats associated with major river basins. The occupation of the site at Tiémassas at the margins of the Saloum delta is a notable exception42, although direct proxies for palaeoenvironments are typically absent. At Ravin Blanc 1, Douze et al.36 question whether the association of crude bifaces with Levallois technologies marks a local post-Acheulean transitional industry, an un-precedented technology that may relate to the Sangoan or simply an early-MSA tradition. Resolving between these alternative patterns of cultural evolution demands the identification of robustly dated Middle Pleistocene MSA assemblages from West Africa. Given the region harbours substantial population diversity, both amongst modern inhabitants43 and Late Pleistocene populations44, evaluating the early MSA in West Africa may help elucidate relationships between biological structure, behavioural diversity and ecological adaptation.The chronology of the West African MSA has only recently begun to come into focus45. Littoral and coastal vegetation in the region includes Guinean mangroves in estuarine zones and halophytic  scrub in dune and coastal plain settings. The modern setting at the site is halophytic scrub situated less than a kilometre from saline marshes and ancient river channels. Today, the region receives an average annual precipitation of 400–500 mm (ref. 46). The presence of Stone Age material at the site was initially reported in 1941 by Mauny and Corbeil, who considered it an open-air surface site. Excavations conducted in 1975 reported a shallow stratigraphy of 25 cm (ref. 47). We have conducted renewed investigations at the site, enabling us to place a large MSA stone tool assemblage within its chrono-stratigraphic and palaeoecological setting. Here we describe the results of investigation at the site designated Bargny 1 and place them within their wider palaeoanthropological context.To explore early-MSA occupations in West Africa, we re-investigated the site of Bargny , located in a quarry 1.9 km from the modern shoreline and approximately 30 km east of Dakar . The site currently lies within the Sudanian vegetation zone Fig. , a transWe investigated the sediment sequence within a quarry pit that had exposed the local bedrock, comprising undulating banded limestones, cleaning the existing section before excavating a 1.75 × 1 m trench in artefact-bearing deposits, to recover lithic artefacts and sediment samples for further analysis (Methods).Six major sediment units were identified in the field and further resolved through laboratory analyses Fig. . The tope) overdispersion and high De reproducibility (Methods and Supplementary Information We recovered five samples for dating from Units 1 (0.45 m), Unit 3 (1.9 m), Unit 4 (2.6 m), the interface of Units 4 and 5 (2.8 m) and Unit 5 (3.1 m). We dated coarse quartz grains with optically stimulated luminescence (OSL) at the single-aliquot level, with all samples possessing good luminescence characteristics, OSL signals dominated by a fast component, low levels of palaeodose   and grass composition  to characterize past vegetation cover at Bargny  and sedge (Cyperaceae), which are common in the brackish wetlands located within 1 km of the site. This agrees with the regional pattern of arid conditions spanning the end of MIS 3 to the onset of the Holocene. Water stress and short-grass indices are tightly constrained around low values in units 4 and 5  and typical brackish mangrove taxa . Supported by the sedimentological results, we interpret the phytolith and pollen signal to indicate that estuarine conditions persisted at Bargny during the latter half of MIS 6, probably supported by local hydrogeological conditions and spring activity, which are common in the modern landscape around the site.Phytoliths from Units 1, 2 and 3 show a stronger signal of water stress, a higher ratio of short- to long-grass phytoliths and are most similar to modern samples from the Sahelian and Saharan vegetation zones  was recovered from Unit 5, which represents all stages of lithic reduction, suggesting on-site activity  and core-on-flake fragments (n = 6), with unipolar, orthogonal and centripetal scars present. Informal core reduction is evident from the presence of multi-platform cores and fragments (n = 27) and single-platform cores and fragments (n = 6). Retouched artefacts are extremely scarce, comprising seven scrapers. This stone tool assemblage is characteristic of both regional and continental expressions of MSA lithic technology.A stone tool assemblage n = 77 was reco53, Haua Fteah55, Ifri n’Ammar56 and Rhafas58), eastern Africa  and southern Africa Fig. . Levallon = 31; Fig. 36 and the Falémé valley37, lower Senegal valley66, Gambia valley37, Ounjougou35, Birimi and Bilma67 and Tiémassas42, our results from Bargny extend the time frame in which a stable and enduring suite of lithic-reduction practices persisted in the region, spanning the late Middle Pleistocene to the onset of the Holocene  to the Holocene that is consistent with records from Bargny 1 and across the region  expansions across the Sahara. Understanding of MSA occupations of forested regions to the south remain poorly known in terms of technology and chronology, limiting the scope of comparisons. The archaeological record supports genetic and fossil records that suggesting longstanding population continuities and limited inter-regional contacts. The absence of demographic pressures in the region could have limited the scope for adaptive innovation74, consistent with the broad technological continuity in the region from the 150 ka occupation of Bargny to the 10 ka occupation of Saxomununya37.The occupation at Bargny around 150 ka highlights that inhabitation of the Senegambian refugia precedes both wider expansions into novel habitats in MIS 5 and particularly across the adjacent arid landscapes of the Sahara75, whereas in tropical Africa, inland sites have been the main focus, even in regions such as eastern Africa that share comparable scales of exploration by Stone Age archaeologists69. Panga ya Saidi, on the Kenyan coast, provides evidence of long-term engagement with coastal resources, although distinct from the more-intensive records from northern and southern Africa34. In West Africa, the occupation of Bargny supplements evidence from Tiémassas42, suggesting repeated inhabitation close to the Senegalese coastline spanning MIS 6–2, yet these records do not yet illustrate clear engagement with coastal resources. Relict populations of Avicennia still occupy brackish mangroves in infrequently inundated backwaters fringing the West African coastline as far as 19° N (ref. 76). Mangroves have high habitat heterogeneity and accessible subsistence resources, making them potential hotspots for Pleistocene forager populations. Adaptation to exploit these resources has the potential to open new axes for population expansions in West Africa77. Accelerating research interest in the West African Stone Age provides the means to test such proposals. As new data emerge, we must consider that from at least the end of the Middle Pleistocene, West Africa may have been a source of behavioural and biological diversity, enabling regionally distinct patterns of human evolution through the Late Pleistocene.The importance of the coastline to Pleistocene populations in tropical Africa has seen highly limited investigation to date. Substantial research has been conducted into patterns of coastal and marine exploitation elsewhere that demonstrate their early importance and integration into wider subsistence patterns. Such studies benefit from extensive research histories on near-coastal cave sitesThe excavation site is located within a quarry, where the sediment sequence had not been removed due to its proximity to a site where mystic rituals and practices of a Lebou community were undertaken. Quarry activities had partially exposed the archaeological horizon, enabling us to identify and target the sediment unit of interest. Following cleaning of any sediment disturbed by quarrying activity, we excavated a 1.75 × 1 m area of intact sediment deposit, differentiating between discrete sediment units subdivided into 5 cm arbitrary horizons to control artefact provenance and recovery. All excavated sediments were wet sieved through a 5 mm mesh to maximize artefact recovery. Cleaning of the entire sediment sequence in a sampling column was undertaken before recovering additional sediment samples for sedimentological, geochronological and palaeoecological analyses, with bulk sediment samples recovered at 5 cm intervals.For laser particle-size analysis, sieved sediment samples  were bathed for 24 hours in sodium hexametaphosphate (4.4%) solution and agitated in an ultrasonic bath, with samples rinsed in purified water before analysis in a Malvern Mastersizer 3000. Characterization of the grain-size results were conducted using Gradistat. For loss on ignition studies, sediment samples (~10 g) were weighed  and heated in a muffle furnace to 105 °C, 550 °C and 950 °C  to calculate the proportions of water, total organic matter, carbonates and mineral residue.78  were considered (Supplementary Table OSL dose rates were based on concentrations of potassium (K), thorium (Th), uranium (U) and Rubidium (Rb) as determined by inductively coupled plasma mass spectrometry and converted to annual dose rates following Guerin and colleagues78 Table . Attenua78 Table , and cosnt ref. 8 were con82. Luminescence measurements of 8 mm in diameter aliquots used a Risø DA-20 luminescence reader. Sample palaeodoses (De) were measured using the single-aliquot regenerative protocol83 with an experimentally derived preheat of 260 °C for 10 seconds. Twenty-four replicate aliquots per samples were measured. Sample De replicates were normally distributed with low overdispersion  before having sands and clays separated by gravity settling and centrifugation–decant cycles at 2,500 r.p.m. for 2 min. At this point, samples were spiked with 87.Macroscopic evaluation of all lithic artefacts established patterns of raw material use after which artefacts were separated into basic technological categories  and identification of fragmented pieces. All specimens were weighed and basic metric attributes recorded . Alternate technological approaches  and blank types  were identified through evaluation of flake-scar characteristics, number and formulation of exploited surfaces, nature of flaking platforms and the direction of exploitation from the flaking platform. Comparative analyses of stone tool assemblages from MIS 6 elsewhere in Africa and throughout the West African MSA identified the presence/absence of 16 stone tool forms, including backing, bipolar technology, blade technology, borers, burins, centripetal technology, large cutting tools, denticulates, Levallois blade technology, Levallois-flake technology, Levallois point technology, notches, platform core technology, point technology, retouched bifacial pieces and scrapers, following refs. Further information on research design is available in the Supplementary InformationSupplementary Information 1: luminescence dating . Supplementary Information 2: plant microfossils . Supplementary Information 3: archaeological comparisons and refugia analysis .Reporting Summary


ObjectiveLumbar fusion performed through lateral approaches is becoming more common. The interbody devices are generally supported by supplemental posterior fixation implanted through a posterior approach, potentially requiring a second incision and intraoperative repositioning of the patient. A minimally invasive lateral interspinous fixation device may eliminate the need for intraoperative repositioning and avoid disruption of the supraspinous ligament. The objective of this in vitrobiomechanical study was to investigate segmental multidirectional stability and maintenance of foraminal distraction of a lateral interspinous fixation device compared to commonly used pedicle screw and facet screw posterior fixation constructs when combined with lumbar interbody cages.MethodsSix human cadaver lumbar spine specimens were subjected to nondestructive quasistatic loading in the following states: (1) intact; (2) interspinous fixation device alone and (3) with lateral interbody cage; (4) lateral lumbar interbody cage with bilateral pedicle screws; (5) lateral lumbar interbody cage with unilateral pedicle screws; and (6) lateral lumbar interbody cage with facet screws. Multidirectional pure bending in 1.5 Nm increments to 7.5 Nm, and 7.5 Nm flexion-extension bending with a 700 N compressive follower load were performed separately with optoelectronic segmental motion measurement. Relative angular motions of L2-L3, L3-L4, and L4-L5 functional spinal units were evaluated, and the mean instantaneous axis of rotation in the sagittal plane was calculated for the index level. Foraminal height was assessed during combined flexion-extension and compression loading for each test construct.ResultsAll implant configurations significantly restricted flexion-extension motion compared with intact (p < 0.05). No significant differences were found in flexion-extension when comparing the different posterior implants combined with lateral lumbar interbody cages. All posterior fixation devices provided comparable neuroforaminal distraction and maintained distraction during flexion and extension.ConclusionsWhen combinedwith lateral lumbar interbody cages, the minimally invasive lateral interspinous fixation device effectively stabilized the spine and maintained neuroforaminal distraction comparable to pedicle screw constructs or facet screws. These results suggest the lateral interspinous fixation device may provide a favorable alternative to other posterior systems that require patient repositioning during surgery and involve a greater disruption of native tissues. The primary goal of lumbar fusion is to permanently stabilize the operated segment, typically in combination with a neural decompression procedure to address various neuropathies. As described in a comprehensive review, this stabilization is best achieved with a combination of interbody fusion and posterior fixation . TraditiLumbar spinal stenosis is a common condition with approximately 1.4 million new cases diagnosed annually in the US . SurgicaSpecimen selection and preparationSix cadaver lumbar spines (L1-sacrum) were acquired for testing from an American Association of Tissue Banks accredited tissue bank. The ages ranged from 40 to 80 years; male to female ratio was 1:2. Donors with a history of spine surgery or abdominal surgery were excluded. Specimens were fluoroscopically screened for any signs of deformity or prior spine surgery before inclusion into the study. The cadaver spinal columns were cleaned of musculature and soft tissue with care taken not to disrupt any ligaments connecting the vertebrae. Each specimen was potted at L1 and S1 with epoxy resin.Device descriptions and techniquesTo accommodate anatomic restrictions of the cadaveric lumbar spines, three sizes of lateral interbody cages  were used in this study. Pedicle screws of 6.5 mm diameter and titanium alloy fusion rods of 5.5 mm diameter were used for the bi-lateral and uni-lateral pedicle screw constructs in this study, and 4.5 mm facet screws were used for the facet screw constructs. The minimally invasive lateral IFD used for the study was the Minuteman G3 ; the second construct was an IFD implanted at L3-4; and the third construct was a direct lateral lumbar interbody fusion (LLIF) cage placed in the L3-4 disc space in addition to the IFD (LLIF+IFD). This group received a unilateral annulotomy and discectomy followed by a contralateral release of the annulus to allow for the distraction of the disc space. The distraction of the neuroforamen for both constructs that included the IFD was achieved by sizing the implant equal to the interspinous distance at the anterior base of the cranial and caudal processes measured on fluoroscopy. To prepare the specimens for this device, a lateral approach rasping of the interspinous ligament and decortication of the cranial and caudal spinous processes was performed. The device was passed through the interspinous ligament, leaving the remaining interspinous ligament and all of the supraspinous ligament intact. After positioning, the extension plates on the distal side were deployed and the device was compressed against the spinous processes of the level being treated. The fourth construct was an LLIF cage in the L3-4 disc space combined with bilateral transpedicular fixation screws at L3 and L4 followed by connecting fusion rods (LLIF+biPS). This group received pilot holes with a standard point awl followed by pedicle screw insertion. No tap was used for pilot hole preparation. Distraction of the neuroforamen was achieved by locking the caudal pedicle screw blockers to the rods, followed by a 2 mm distraction of the cranial screws using a standard distractor instrument. The fifth construct included the interbody cage in the L3-4 disc space in addition to unilateral trans-pedicular fixation screws at L3 and L4 with a connecting fusion rod (LLIF+uniPS). A similar 2 mm distraction was applied to the single fusion rod and screw construct. The sixth construct included the interbody cage in the L3-4 disc space in addition to trans-facet fusion screws between L3 and L4 (LLIF+FS). This group received a pilot hole using a powered drill bit in a caudal-lateral direction from the inferior facet of L3 to the superior facet of L4. Fluoroscopic images of the five instrumented constructs  are shown in Figure Mechanical testingTwo experimental loading protocols were conducted using an MTS MiniBionix 8521 testing system . In the first protocol, pure bending moments were generated using custom loading fixtures attached to the MTS system. Multi-directional segmental motions were evaluated under flexion, extension, left and right lateral bending, and axial rotation loading. Bending moments were increased in 1.5 Nm increments to 7.5 Nm. A three-dimensional (3D) motion tracking system  used markers that were attached to L2, L3, L4, and L5 vertebral bodies to track their positions and orientations during loading. Relative angular motions of L2-L3, L3-L4, and L4-L5 functional spinal units were subsequently and concurrently calculated alongside the mean instantaneous axis of rotation in the sagittal plane, which was calculated for the index spinal units between the maximum applied moments.In the second protocol, 7.5 Nm of flexion and extension bending moments were applied with a 700 N axial compressive follower load in accordance with previous studies -13. ForaTo highlight the important observations from the study, the results presented in this article include the primary motions, instantaneous axis of rotation, and foraminal height outcomes from the index level only. Additional data and results can be obtained from the authors upon request.Multi-directional range of motionMean flexion-extension motion at L3-4 was significantly less in all conditions involving an implant compared with INTACT There were no statistically significant differences in the anterior-posterior location of the mean IAR when comparing surgical constructs Figure . In the Foraminal heightThere were no statistically significant differences in foraminal height between any of the constructs when compared to the INTACT specimen or each other Table . There wIn this biomechanical human cadaveric study, a minimally invasive lateral IFD combined with a lateral interbody cage was found to provide segmental spinal stability comparable to that of other constructs employing pedicle screws or facet screws combined with lateral interbody cages. It was also found that foraminal distraction using this interspinous fixation technique was similar to commonly used posterior constructs and that foraminal height was maintained during motion under combined compression with flexion-extension bending moments.The results of the current study are in line with previous investigations; when used with an interbody fusion cage, interspinous fixation produces similar and even superior stability compared to adding pedicle or facet fixation -18. FogeFew biomechanical studies have investigated the ability of interspinous fixation devices to distract the neuroforamen. Lazaro et al.  investigThe results of the current study found that the laterally placed interspinous fusion device combined with a lateral interbody fusion cage provided multi-directional segmental stability and maintenance of neuroforaminal distraction during flexion-extension loading similar to pedicle screws or facet screws used with lateral interbody fusion cages. Thus, from a biomechanical standpoint, the laterally placed interspinous fixation device is a viable alternative to currently used posterior fixation devices used with lateral interbody cages. The lateral IFD implant in this investigation retains the supraspinous ligament, which may increase stabilization as compared to other interspinous devices that remove or otherwise disrupt this ligament during placement. Likewise, there is penetration through a portion of the interspinous ligament with the IFD procedure rather than the larger disruption of this ligament during implantation of other minimally invasive interspinous devices. The benefit of the preservation of these ligaments for joint stabilization appears to be supported by a biomechanical study that suggests that the supraspinous-interspinous ligament complex contributes more than a third of the resistance to flexion in the intact joint .Limitations of this study include using a cadaveric model, which undergoes ambient degradation between the rounds of testing that is unlikely to be observed in an in vivo study. The number of human cadaveric spine specimens was also a limitation of this study. While using a much larger number of specimens would enable minimization of environmental degradation and minimize or quantify prior-loading degradation (intra-specimen variability), the use of the study design employed in this study - testing each construct on each spine specimen - reduces the amount of inter-specimen variability in the data and significantly reduces the number of human spine specimens required to gather an equal number of data points. The loading was non-destructive and the specimens were kept moist; however, performing successive tests on the same specimen could result in lower apparent stability in the later tests performed. The experiments were conducted on lumbar spines with a male-to-female ratio of 1:2 and ages ranging from 40 to 80 years. Therefore, it may not be appropriate to generalize the results from this study to different demographics or patients with varying initial spinal conditions. Additional studies are required to determine if the joint stabilization demonstrated in the in vitro environment will correspond to comparable stabilization and satisfactory levels of bony fusion occurring in vivo. While maintenance of foraminal height under combined compressive and flexion-extension loading was demonstrated in this study, increases and maintenance of neuroforaminal area and canal area were not assessed, but would be relevant in evaluating the clinical utility of posterior fixation devices used without direct neural decompression, in particular, the lateral IFD, in spinal stenosis cases. Although the subsequent addition of posterior instrumentation is common practice in cases of subsidence or other complications related to stand-alone lateral interbody cages, the competence of posterior fixation constructs in combination with LLIF cages to resist cage subsidence cannot be ascertained from these biomechanical study data. Finally, a clinical investigation is warranted to determine if the IFD fulfills the potential to reduce operative time and the need for additional incisions by avoiding the placement of supplemental fixation from a posterior approach following the placement of an interbody fusion cage from a lateral approach.When combined with LLIF, the minimally invasive lateral interspinous fixation device effectively stabilized the spine and maintained neuroforaminal distraction comparable to pedicle screws and rods or facet screws. These results suggest the lateral interspinous fixation device may provide a favorable alternative to other posterior fixation systems that require patient repositioning during surgery and involve a greater disruption of native tissues. Clinical investigations are needed to determine the benefits of lateral interspinous fixation combined with lateral interbody fusion with regard to operating time, rates of subsidence, levels of bony fusion, and other clinical outcomes.


Cell, Hasegawa and colleagues reveal that cytotoxic CD4-positive cytotoxic T lymphocytes (CD4+ CTLs) eliminate senescent fibroblasts with a surface expression of the immunologically important human leukocyte antigen class II (HLA-II) and the human cytomegalovirus glycoprotein B (HCMV-gB).1 This implies that a virome-immune cell interaction controls the fate of human skin cells that are linked to the aging process and associated diseases.In a recent publication in INK4A and p21CIP1) attenuate these processes and are biochemical markers of senescent cells. Notably, senescent cells are not inert but contribute to pathological functions in inflammation, cancer, age-related diseases, and other ailments.A previously unknown rise in life expectancy of current human societies strengthens the desire to uncouple the aging process from age-associated risk factors and chronic disorders. The persistence of old and damaged cells in tissues hampers this goal. Cells with such a senescent phenotype carry typical morphological and biochemical alterations. For example, cyclin-dependent kinases induce cell cycle progression for cell growth and renewal. Cyclin-dependent kinase inhibitors  compared to older skin tissues (n = 31) had less p16INK4A-positive and p21CIP1-positive senescent fibroblasts  and old skin (53–74 years). Fibroblasts are the main cell type of our connective tissues. As anticipated, younger skin tissues  occurs with a prevalence between 60-100% worldwide.+ CTLs  valproic acid.3 HDACi have been intensively tested in clinics and some have been approved for the treatment of blood cancers. These drugs evolved from evolutionary old endogenous and microbial compounds .4 Whether the intake of such agents can modulate interactions of infected host cells and CD4+ CTL and if this exceeds disadvantageous side-effects is unknown. The still open mechanisms through which HCMV-gB levels rise in senescent cells may likewise provide opportunities to stall aging processes.The very high prevalence of HCMV in humans may still offers an avenue to target infected senescent cells—through boosting CD45 Hormonal changes—alone or in conjunction with HCMV and immune cells—might have contributed to the significant delay in a further accumulation of senescent cells in this age group.Hasegawa and colleagues analyzed only female skin samples. A missing sex tropism of HCMV makes it very likely that male skin behaves similarly. As skin samples were surgery leftover material, the donors’ health states may have impacted the number of senescent cells. It additionally needs consideration that the older skin group was from individuals aged over 50 years, suggesting menopausal life. Estrogen and testosterone critically impact human aging processes.In sum, Hasegawa and colleagues provide human data supporting a new framework for the interactions between viruses and their hosts’ immune systems.