Lepidochelys kempii) and loggerhead (Caretta caretta) turtles strand during fall on the beaches of Cape Cod , with total stranding numbers sometimes exceeding 300 turtles per year. Once rehabilitated, turtles must be released at beaches with appropriate water temperatures, often requiring transportation to southeastern coastal states of the USA. These transportation events (transports) may approach or exceed 24 h in duration. Kemp’s ridley turtles are known to exhibit an adrenal stress response during such transports, but the effect of transport duration has been unclear, and no other sea turtle species has been investigated. To assess whether transport duration and/or species affects physiological reactions to transport, we studied pre- and post-transport physiological measures in Kemp’s ridley and loggerhead turtles transported by ground for <6, ∼12, ∼18, or ∼24 h, comparing with matched “control events” in which turtles were studied without transport. Blood samples were analyzed for four stress-associated measures  and nine measures of clinical status . In both species, stress-associated measures elevated significantly during transport, while handling without transport had no significant effects. Loggerheads exhibited a greater stress response than Kemp’s ridleys across all transport durations. These results indicate that sea turtles do react physiologically to ground transport; therefore, minimizing transport time and streamlining transport logistics (where feasible) may help ensure release of rehabilitated turtles to sea in the best possible condition. Nonetheless, both species remained in good clinical condition even after 24 h transport, indicating that current transport protocols are generally safe for sea turtles from a clinical perspective.Many juvenile Kemp’s ridley ( Lepidochelys kempii, “Kemp’s ridleys”), a critically endangered species , a vulnerable species, the next most common. The stranded Kemp’s ridleys and loggerheads are usually found “cold-stunned,” a hypothermia-like state entailing low body temperature and related clinical abnormalities that include electrolyte derangements, dehydration, acidosis, pneumonia, sepsis, and injuries  typically occur by ground in small trucks or vans, with the turtles out of water in individual padded crates or boxes. Transport durations may range from <6 h  to >24 h (to Florida beaches), depending on calendar month, local water conditions at potential release sites, and other logistical constraints , 2019. ISome stress is inevitable when transporting wildlife . Ground In prior studies, we confirmed that Kemp’s ridley turtles do experience an adrenal stress response during ground transports of 13 and 26 h duration, with pronounced and significant elevations in plasma corticosterone, plasma glucose, WBC, and H/L ratio immediately post-transport when compared with pre-transport , 2019. C2, pCO2, HCO3 [bicarbonate], sodium, potassium, calcium, lactate, and hematocrit) that are commonly used to monitor clinical status, following In this study, we sought to fill this information gap by evaluating physiological stress responses of juvenile Kemp’s ridley and loggerhead turtles transported for four different durations , representing typical transport durations to four commonly used release sites along the US eastern seaboard. Our study design relied on opportunistic sampling of turtles already scheduled for transport. We compared pre- and post-transport measures of four “stress-associated” variables identified in prior studies  rehabilitation clinic in Quincy, MA, USA. Transport of any individual turtle occurred the spring or summer after that turtle’s initial date of stranding, with all turtles transported <10 months after stranding. Kemp’s ridley turtles in this study were estimated to be 2–3 years of age , in compliance with guidelines of the Animal Care and Use Committees of the NEAq  and Northern Arizona University (IACUC protocol 16-007). Turtle transports and beach releases were performed in compliance with all applicable local, state, and federal regulations and guidelines.n = 8 turtles per species per transport duration bin.Our experimental design involved opportunistic study of Kemp’s ridleys and loggerheads that were scheduled for transport to four frequently used release locations: local Massachusetts beaches , the Chesapeake Bay area , the coast of Georgia (∼18 h transport), or the coast of northern Florida (∼24 h transport). Transports were thus grouped into four duration bins of <6, ∼12, ∼18, and ∼24 h, with actual transport durations expected to fall within ±2 h of the target transport time. Unpredictable traffic conditions and weather sometimes cause a transport to deviate by ∼1–2 h from the planned transport time. Our planned sample size, based on variation observed in pilot data, was Transport events were compared with data from matched control events during which turtles were handled and sampled twice, but with no transport (details below). Control events occurred 2–3 weeks prior to the matched transport event, with the control event matching the scheduled transport event for estimated duration, time of day, species, and number of turtles. Where possible, the same turtles were used for a matched control event and a transport event (same duration), but it was not always possible to study the same turtles for the control event. No turtle was studied for multiple events of the same type and duration, i.e., pseudoreplication did not occur.At the initiation of each control event or transport event, turtles were removed from their pools at the NEAq sea turtle rehabilitation clinic in Quincy, MA, USA, and a 2–3 mL heparinized blood sample (“pre” sample) was immediately collected from the external jugular vein as previously described , 2009, fAfter sampling and examination, turtles scheduled for transport were placed into individual padded crates with air holes, given subcutaneous fluid therapy , and coated lightly with water-soluble lubricant to reduce dehydration, following the NEAq clinic’s routine transport protocols. Crates were closed and loaded into the rear cargo area of large sport utility vehicles with ambient temperature set to 26°C, the same temperature as the NEAq holding tanks. Vehicle temperature was monitored continuously with digital remote temperature probes placed in the rear cargo area near the turtles. For <6 and ∼12 h transports, pre-transport sampling occurred in the morning, with post-transport sampling occurring later on the same day, i.e., these transport durations did not include an overnight phase. The ∼18 and ∼24 h transports were overnight events, with pre-transport sampling occurring in the morning (∼24 h transports) or afternoon (∼18 h transports), driving through the night, and post-transport sampling occurring the following morning. In all transport events, efforts were made to minimize potential disturbance to turtles, streamline logistics, and minimize driving durations. Brief stops (∼15 min) for refueling and driver-change occurred approximately every 3–4 h.For control events, turtles were removed from tanks and re-sampled (“post” sample), as described above, after the designated event duration . For transport events, upon arrival at the destination, turtles were unloaded from vehicles and carried (still in closed crates) to a shaded area for sampling. The transport crate was then opened, with the moment of opening of the crate designated as “time zero” for measurements of bleed time. A second heparinized blood sample was immediately taken (“post” sample) and a second clinical examination performed using the methods described above, with results compared with pre-transport samples and to control events.g for 5 min, with plasma pipetted to a vapor-proof cryovial and shipped on dry ice to the endocrine laboratory of the Buck Laboratory, Northern Arizona University, Flagstaff, AZ, USA, for corticosterone assay (details below).Our analytical protocols are described in detail in ® 1 Analyzer Model 300A . Two i-STAT machines were used; each turtle’s two samples were always analyzed by the same i-STAT machine. Generally, data appeared comparable across machines (data not shown).Immediately upon collection of the sample, whole blood was loaded into both an i-STAT CG4+ Test Cartridge and an i-STAT CG8+ Test Cartridge, which were analyzed using a portable battery-powered handheld point-of-care analyzer, VetScan i-STAT2, pCO2, HCO3 (bicarbonate), and lactate. The CG8+ cartridges measured sodium, potassium, ionized calcium (iCa), and glucose, and also produced backup data on pH, pO2, pCO2, and HCO3. Data for pH, pO2, pCO2, and HCO3 were taken from the first cartridge analyzed, which in almost all cases was the CG4+ cartridge. In three cases , the CG4+ cartridge initially failed and hence the CG8+ cartridge was then analyzed first while a new CG4+ cartridge was prepared, in order to minimize delays in obtaining blood gas measurements. The pH, pO2, and pCO2 data were all temperature-corrected for that turtle’s cloacal temperature, and iCa data were pH-corrected using previously described formulas  . Descriptive statistics (median and mean±SEM or SD as appropriate) were used to summarize stress-associated and clinical health measures for both species, as well as actual transport durations, bleed time, body mass, and carapace measurements. Absolute data on corticosterone concentration, WBC, H/L ratio, and lactate concentration were log-transformed to adjust for non-normal distribution based on visual inspection of residual distributions and Q–Q plots. Lactate data were frequently below detectability limits (see the “Results” section) and for statistical analysis were assigned a nominal value of one-half the detectability limit. Variables were then divided into two multivariate datasets for analysis , which are widely used to monitor clinical health. For stress-associated and clinical variables, we calculated the change in measured levels from the pre-transport sample  to the post-transport sample for each turtle (paired before-after data). Resulting data represented the physiological response of turtles to experimental events, and better enabled exact comparisons by adjusting for any inherent differences in baseline physiology of turtle species. Physiological data were analyzed using multivariate linear mixed model (LMM) frameworks with normal probability distribution and identity link function. For each of the two datasets , we conducted a series of LMM analyses that (1) determined the influence of control sampling (using pre- and post-event absolute data from control experiments for each species); (2) compared physiological responses of both species across the different event scenarios ; and (3) examined data for each species independently to better determine how different transport durations affect physiological responses in Kemp’s ridley and loggerhead turtles, when compared with responses from control events. A full factorial mixed model allowed both categorical fixed factors  and random components  to be potentially fitted in analyses. Body mass was included as a covariate in all models, as differences in body condition and/or age could affect responses to environmental stressors  for any clinically relevant deviations from expected values for healthy individuals, when compared with data reported for juveniles of these species  at a significance level of P < 0.05.Parallelism of the corticosterone assay for loggerhead plasma was assayed with an is as in , 2019: .The corticosterone RIA exhibited good parallelism for loggerheads, with no significant difference in slope of the binding curves of serially diluted loggerhead plasma pool and corticosterone standards : <6 h events, 3.96 h ± 1.29 h (mean±standard deviation), range 2.3–5.9 h, F4,61=0.73, P = 0.58; clinical data, F9,52=0.73, P = 0.68; loggerheads: stress data, F4,49=0.59, P = 0.67; clinical data, F9,40=1.22, P = 0.31). When control-event variables were examined individually, P > 0.05 for all measured variables except for iCa of loggerheads   . These rF4,133=30.12, P < 0.001; P < 0.001; F9,113=2.00, P = 0.05; In contrast, transportation was found to markedly and significantly influence the physiological stress response of both sea turtle species  and glucose concentrations  were significantly elevated above control levels after even the shortest transport duration (<6 h), and both measures showed similar response levels across all transport durations, i.e., corticosterone and glucose appeared to respond maximally even at short transport durations, with no additional significant elevation at longer durations , with loggerheads exhibiting a more pronounced physiological stress response than Kemp’s ridleys in every one of the four stress-related measures  across all transport durations . There was no overlap in body mass between individuals of the two species in this study. Loggerhead mean body mass was 31.7 ± 14.1 kg (±SD) (range 10.0–81.0 kg), mean SCL was 56.5 ± 9.0 cm (range 38.2–84.5 cm), and mean SCW was 48.3 ± 7.1 cm (range 34.2–66.3 cm). Kemp’s ridley mean body mass was 4.65 ± 1.30 kg (range 2.30–8.25 kg), mean SCL=30.7 ± 3.1 cm (range 24.0–38.1 cm), and mean SCW was 28.6 ± 3.2 cm (range 22.4–37.4 cm). Differences in body mass of turtles in this study did not significantly influence the observed physiological responses to transportation .As expected, loggerheads were significantly larger and heavier than Kemp’s ridleys , with a median bleed time for loggerheads of 2.35 min (range 0.65–18.23 min) when compared with median bleed time for Kemp’s ridleys of 1.87 min (range 0.63–10.25 min). However, there was no apparent relationship of bleed time to corticosterone in either species , i.e., bleed times were rapid enough in both species that corticosterone levels were unlikely to be affected by handling. In total, combining both species, bleed times were <3 min for 258 samples (78.9% of samples), <5 min for 305 samples (93.3%), and <10 min for 324 samples (99.1%), with only three samples exceeding bleed times of 10 min. The latter three samples include a Kemp’s ridley post-transport sample taken at 10.25 min after initial disturbance, a loggerhead post-transport sample taken at 12.95 min, and a loggerhead post-control sample taken at 18.23 min, the delays in all three cases due to difficulty obtaining blood. Even these three samples had corticosterone concentrations typical for the turtle’s species and experimental group. Given lack of evidence for any effect of bleed times in this study on corticosterone concentration, all corticosterone data were retained in the final dataset. Additional sample timing data, along with vital rates and detailed CBC data, are reported in the Bleed times were slightly, but significantly, longer for loggerheads than for Kemp’s ridleys , H/L ratio , or corticosterone  due to hemolysis or unanticipated shipping delays. These sporadic cases of missing data were approximately equally distributed across species, event types, and event durations.Final sample sizes of turtles are shown in Kemp’s ridley and loggerhead turtles both show evidence of a consistent physiological stress response due to ground transport, i.e., significant elevations in four stress-associated variables  after transports of at least ∼6 h (loggerheads) or 12 h (Kemp’s ridleys). Comparison to matched control data indicates that these changes are not attributable to effects of handling, repeated sampling, food deprivation, or time of day, but appear specifically due to the stimuli of transportation  Such physiological responses to transport have been documented before in Kemp’s ridleys, but until now, no information on transport-related stress was available for other sea turtle species. Our data demonstrate that loggerheads also respond to transport, and, by extension, suggest that other sea turtle species may be affected by transport-related stress as well.Species differences in physiological responses were evident, with loggerheads exhibiting more pronounced physiological changes at every transport duration than did the smaller Kemp’s ridleys. Even at the shortest transport durations tested (<6 h), some stress measures of loggerheads exceeded the 24 h maxima recorded in Kemp’s ridleys. Furthermore, loggerheads seemed to attain a maximum or near-maximum stress response at relatively shorter transport durations, with several stress-related variable measures elevating markedly and significantly at the shortest duration examined (<6 h) and plateauing thereafter. In contrast, Kemp’s ridleys showed milder changes that tended to increase incrementally with longer transport durations. It is possible that some of these differences may be due to species-specific variation in absolute concentrations of corticosterone, sensitivity to corticosterone, and, in general, clinical significance of these measures.Overall, however, our loggerhead results, when compared with published data on stress physiology in these species , suggestFor Kemp’s ridleys, only the more prolonged transport durations of at least 12–18 h caused significant increases in glucose, corticosterone, and H/L ratio, while WBC counts did not significantly elevate until 24 h. Comparisons to published literature on this species suggest that transportation did not induce a maximum physiological stress response in Kemp’s ridleys, even after 24 h of transport . FollowiImportantly, despite the significant effect of transport on stress-related measures, other clinical measures of health  remained within acceptable limits for both species and the turtles completed transport in good condition. Nonetheless, in the absence of data to the contrary, we suggest erring on the side of caution when transporting loggerheads (and perhaps other larger-bodied sea turtles), i.e., minimizing transport duration when possible.Recent analyses have suggested a potential causal relationship between ongoing increases in sea surface temperature and the simultaneous increases in turtle stranding numbers on Cape Cod . The numHowever, the documented negative long-term effects of elevated corticosterone in other species warrant continued attention to this issue. We suggest that in the absence of data on potential long-term effects of corticosterone in chelonians, it would be advisable to minimize transport stress where possible. In our experience, transport duration, and presumably associated stress, can be considerably minimized with attention to the logistical details of a sea turtle transport event. Feasible and effective changes to reduce transport time from the NEAq clinic have included: scheduling additional staff to reduce pre-transport preparation time, scheduling transports to reduce chances of encountering heavy traffic at major cities en route, eliminating meal stops, shortening refueling stops, scheduling sufficient additional staff or vehicles to enable driving throughout the night, and encouraging efficient use of time for any necessary media, public-relations, and research activities during subsequent beach releases. Our pre-transport preparation protocols  may also be a factor in promoting good clinical condition even during longer transports. The transport environment, too, is likely important; our routine protocols include placing turtles in padded, dim, individual containers; minimizing handling, conversational noise, vehicle noise, and unnecessary acceleration/deceleration; usage of dim lighting in turtle cargo areas; and continuous monitoring and adjustment of cargo-area temperature. While our study did not specifically test the effect of these factors, we consider it likely that they help minimize stress. We note, also, that it is possible that same-day transports might have differing physiological effects than overnight transports (the present study was not designed to separate effects of an overnight phase from transport duration). Overall, we recommend that clinicians and veterinarians continue to study and adjust sea turtle transport protocols so as to achieve maximum health and minimal physiological stress for a given transport duration, with the ultimate goal of releasing sea turtles to sea in ideal or near-ideal condition. Future studies should seek to characterize short-term and long-term post-release behavior and outcomes after various transport events, to enable further refinements in transportation protocols.obaa012_Supplementary_DataClick here for additional data file.


The difference of flowering times in 9 years’ field tests was large in “Khao Nam Jen” (36.7 days) but small in “Koshihikari” (9.9 days). Part of this difference was explained by two QTLs. A CSSL with a “Khao Nam Jen” segment on chromosome 11 showed 28.0 days’ difference; this QTL would encode a novel flowering-time gene. Another CSSL with a segment from “Khao Nam Jen” in the region around Hd16 on chromosome 3 showed 23.4 days” difference. A near-isogenic line (NIL) for Hd16 showed 21.6 days’ difference, suggesting Hd16 as a candidate for this QTL. RNA-seq analysis showed differential expression of several flowering-time genes between early and late flowering seasons. Low-temperature treatment at panicle initiation stage significantly delayed flowering in the CSSL and NIL compared with “Koshihikari”. Our results unravel the molecular control of flowering time under ambient temperature fluctuations.Climate resilience of crops is critical for global food security. Understanding the genetic basis of plant responses to ambient environmental changes is key to developing resilient crops. To detect genetic factors that set flowering time according to seasonal temperature conditions, we evaluated differences of flowering time over years by using chromosome segment substitution lines (CSSLs) derived from  Oryza sativa L.) originated from a wild rice species (Oryza rufipogon Griff.), which grows mainly in the tropics [Global warming is likely to reach 1.5 °C between 2030 and 2052 if it continues at the current rate [ tropics . At the  tropics . There iOsGI–Hd1–Hd3a pathway and the Ghd7–Ehd1–Hd3a/RFT1 pathway. Both pathways are regulated by light perception, daylength and the circadian clock. These previous molecular genetic studies show the importance of response to daylength in the determination of rice flowering time.Daylength is a primary environmental factor that determines maturation (harvest) time through induction or suppression of flowering and consequently adaptability to each growing area in plant species ,8. Rice Brassica vegetables [PhyB, PIFs, DELLA and the evening complex, which includes ELF3 [indica and japonica cultivars [Ehd1, Hd3a and RFT1 was repressed under long daylength and low temperatures. The data suggest that temperature response is a decisive factor in determining flowering time in rice. However, despite their importance in relation to breeding programs, the molecular mechanisms involved in temperature response remain largely unknown. It is necessary to elucidate the genetic mechanisms underlying the determination of flowering time in response to ambient temperature in rice.Temperature is another important ambient environmental factor that determines flowering time. Vernalization (low-temperature) treatment promotes flowering in several cereal and horticultural species such as wheat, barley and getables ,15. In Ades ELF3 ,18,19,20des ELF3 ,22,23. Fultivars . In addiultivars ,26. Theyjaponica rice cultivars “Koshihikari” and “Khao Nam Jen”. “Khao Nam Jen” had a large difference. Among the 40 CSSLs, two lines with a “Khao Nam Jen” segment on chromosomes (Chrs.) 11 and 3 (the latter including the Hd16 region) had over 15 days’ difference. The QTL on Chr. 11 would be a novel flowering-time gene, because no allelic differences have been found in genes previously isolated in this region. Low-temperature treatment at the panicle initiation stage delayed flowering time in both lines. RNA-seq analysis revealed that expression of the flowering-time genes Ehd1, Hd3a and RFT1 was shifted later in the flowering season in the CSSL than in “Koshihikari”. Through these analyses, we detected novel genetic factors involved in the control of flowering time under different ambient temperatures during the rice-growing season.Here, we evaluated differences of flowering time over 9 years (2011 to 2019) in Asian rice cultivars and chromosome segment substitution lines (CSSLs) derived from crosses between We found wide variations in flowering time  of 18 Asian rice cultivars during 2011 to 2019 . For exa4F6 generation , we carried out RNA-seq analysis in 2015 (early-flowering year) and 2016  in “Koshihikari”, 2.4 times in Koshihikari Hd16-NIL and 2.8 times in SL2833  and low temperature (24/20 °C) on flowering in “Koshihikari”, Koshihikari n SL2833 . Low temHd16—are associated with the response of flowering time to ambient temperatures during the rice-growing season.Even though classical genetic studies suggest the importance of temperature response and photoperiod response in determination of flowering time in rice ,22,23, fRCN1 and RBS1 that encode phosphatidylethanolamine-binding protein and heterogeneous nuclear ribonucleoprotein R-type protein, respectively. However, the sequences of RCN1 and RBS1 were identical between “Khao Nam Jen” and “Koshihikari” and expression levels of both did not differ significantly in our RNA-seq dataset in 2015 and 2016. Therefore, this QTL may be a novel flowering-time gene in rice. Further genetic studies such as fine-mapping experiments are necessary to isolate the gene responsible for this QTL.The QTL for flowering time on the short arm of Chr. 11 was detected in only 4 out of the 9 years, with inconsistent directions of additive effects. The flowering time of SL2833, with the “Khao Nam Jen” allele at this QTL, was negatively correlated with maximum temperature in July in all 9 years; flowering was delayed in 2017 (low maximum temperature) and promoted in 2018 (high maximum temperature). Flowering time of SL2833 was significantly delayed under low temperature condition. Therefore, this QTL is associated with responsiveness to ambient temperature in regulating flowering time in rice. It was localized to the region containing Hd16, which was isolated as a flowering time-gene involved in photoperiod sensitivity in rice [Hd16 NIL had a wide difference of flowering time and a large CV among the 9 years. Additionally, low-temperature treatment delayed flowering time in Koshihikari-Hd16 NIL. Our results indicate that Hd16 is associated with both photoperiod sensitivity and response to ambient temperatures. Hd16 encodes casein kinase-I; the “Koshihikari” allele has one non-synonymous substitution in the conserved kinase domain, which results in deficient function, causing decreased photoperiod sensitivity [Hd16 allele from “Khao Nam Jen” promoted both strong photoperiod sensitivity and strong response to ambient temperatures in comparison with the non-functional “Koshihikari” Hd16 allele. Biochemical characterization indicated that a functional Hd16 recombinant protein specifically phosphorylated the proteins encoded by rice flowering-time genes Ghd7 and OsPRR37 and the SLR1 protein in the gibberellin signaling pathway in rice [Hd16 alters the response of flowering time to ambient temperatures through both the photoperiod-sensitive flowering-time pathway and the gibberellin-dependent growth-regulatory pathway.The QTL on the long arm of Chr. 3 corresponds to  in rice ,32,33. Ssitivity . The fun in rice ,32,33. TSeveral other CSSLs had moderately large flowering time fluctuations among the 9 years . SL2809,OsGI–Hd1–Hd3a pathway and the Ghd7–Ehd1–Hd3a/RFT1 pathway. Both pathways promote flowering under short daylength but repress it under long daylength —in each genome [VRN1, which encodes an AP1-like MADS-box transcription factor, is orthologous to OsMADS14, OsMADS15 and OsMADS18 in rice [VRN2, with a zinc-finger motif and a CCT domain, seems to be homologous to rice Ghd7 [VRN3 is homologous to FT-like genes such as Hd3a and RFT1. These studies suggest that homologs of rice photoperiod-sensitivity genes are associated with vernalization response in wheat and barley. Therefore, the genetic pathway that controls the response of flowering time to ambient temperatures might be conserved among cereal crop species.Extensive studies in model plant species have elucidated the details of the flowering-time genetic networks. Six major pathways have been reported in Arabidopsis: the photoperiod, vernalization, gibberellin, autonomous, age and ambient temperature pathways ,35. Efforeviewed . In Arabreviewed ,37. Arabh genome ,39. Whea in rice . VRN2, wice Ghd7 . VRN3 isPhyB, PIF4, YUC8, DELLA and ELF3 [PhyB functions as a thermo-sensor in leaf epidermis and regulates PIF4 activity and the auxin signaling pathway in response to ambient high temperature, as well as to light signals [High temperature accelerates plant growth and development in Arabidopsis through a signaling pathway that includes and ELF3 ,18,19,20 signals . In our  signals . To reveGlobal warming is causing climate change with large temperature fluctuations, in addition to variations in solar irradiation, precipitation and soil moisture, in rice-growing regions ,4. TempeHd16, on the log arm of Chr. 3 and the other QTL, on the long arm of Chr. 11, might be a novel gene. Low temperature treatment at the panicle initiation stage delayed flowering in the two CSSLs with these QTLs. These results provide insights into thermo-sensitive flowering-time regulation in rice and will facilitate our understanding of the molecular basis of the control of flowering time variation under ambient temperature fluctuations and allow the development of climate-resilient crops that are adaptable to temperature fluctuations under climate change.We found a wide difference of flowering time fluctuations among 9 years in Asian rice cultivars. We detected two QTLs in CSSLs with “Khao Nam Jen” chromosome segments in the “Koshihikari” genetic background. One QTL corresponds to indica, 3 aus, 7 japonica), mainly from Asia, were selected on the basis of their geographical origin, cluster analysis of genome-wide genotype data and variation in DTH from a representative rice core collection [4F6 generation, covering 95.9% of the “Khao Nam Jen” genome. Two near-isogenic lines (NILs) in the “Koshihikari” genetic background were developed in previous studies [We selected 18 rice cultivars: “Qui Zhao Zong”, “Davao 1′, “IR64′, “Touboshi”, “Deng Pao Zhai”, “Naba”, “Bei Khe”, “Bleiyo”, “Tupa 121-3′, “Kasalath”, “Muha”, “Koshihikari”, “Nipponbare”, “Basilanon”, “Khao Mac Kho”, “LAC 23′, “Khao Nam Jen” and “Nona Bokra”. These cultivars , Tsukuba, Japan , in 9 years (2011 to 2019). Month-old seedlings were transplanted in mid-May at one per hill in plots with a double row for each line, with 18 cm between plants and 36 cm between rows. The mean daylengths were 12.3 h in April, 13.4 h in May, 14.3 h in June, 14.4 h in July, 13.6 h in August and 12.6 h in September. Cultivation management followed the standard procedures used at the Institute of Crop Science. DTH of individual plants were scored as the number of days from sowing to the appearance of the first panicle in each of the 5 or 24 plants and mean values were calculated. The difference of flowering time of each line was calculated as the difference between the earliest and latest flowering times among the 9 years. The coefficient of variation (CV) was calculated as standard deviation ÷ average flowering time in each year.The difference of flowering time and CV among years in CSSLs and NILs were compared with those of “Koshihikari” by using Dunnett”s multiple comparison procedure in JMP v. 11.0.0 software. For QTL detection in the CSSLs, we used a total of 377 SNP and 112 SSR markers showing polymorphisms between “Koshihikari” and “Khao Nam Jen” reported by the previous studies ,30. QTLsHd3a, RFT1, Ehd1 and Rice ubiquitin2 (UBQ) were measured according to a SYBR Green-based method by using gene-specific primers.We extracted mRNA from the highest fully expanded leaf of “Koshihikari” and SL2812 (“Khao Nam Jen” segment on long arm of Chr. 3) at 2 to 20 weeks after transplanting in 2015 and 2016. Sequencing libraries for RNA-seq analysis were constructed by using the QuantSeq 3′ mRNA-seq Library Prep Kit  with barcodes for Illumina sequencing according to the manufacturer’s instructions. The libraries were sequenced on a HiSeq 4000 platform  using the TruSeq SBS Kit  and the paired-end sequencing method to obtain two 100 bp sequences. Individual reads were trimmed by cutting bases from the start and end of reads if quality ≤20 in Trimmomatic v. 0.36 software and by removing reads with final read length <50 in FASTQC v. 0.11.9 software. Trimmed reads were mapped on the IRGSP1.0 rice genome sequence and gene loci in RAP-DB . Each gehttp://www.naro.affrc.go.jp/org/niaes/aws/). We collected maximum, minimum and average values of air temperature, relative humidity, precipitation, wind speed, wind direction, duration of sunshine, amount of insolation and soil temperature each month from April to November. Pearson’s correlation coefficient was used to assess relationships among DTH and the weather data in JMP.Weather data at the paddy field from 2011 to 2019 were collected from the Weather Data Acquisition System of the Institute for Agro-Environmental Sciences, NARO  were grown in a controlled-environment cabinet under a short daylength (10 h light/14 h dark) or a long daylength (14.5/9.5 h). Relative humidity was maintained at 60% under a photosynthetic photon flux density of 500 µmol m−2 s−1 provided by metal halide lamps that covered the spectrum from 300 to 1000 nm. Ambient temperatures (light / dark) were 30/24 °C, 27/24 °C , or 24/20 °C. DTH in 10 plants of each line was scored and mean values were calculated for each line.“Koshihikari”, Koshihikari


Esophageal candidiasis (OC) is a common AIDS-defining opportunistic infection. Antiretroviral therapy (ART) reduces the occurrence of OC and other opportunistic infections among persons living with HIV (PLHIV). We sought to determine and compare the prevalence of OC in the ART and pre-ART era among PLHIV in sub-Saharan Africa (SSA).We searched PubMed, Embase, Web of Science, and the African Journals Online databases to select studies in English and French reporting the prevalence of HIV-associated OC in SSA from January 1980 to June 2020. Reviews, single-case reports, and case series reporting < 10 patients were excluded. A random-effect cumulative meta-analysis was performed using STATA 16.0, and trend analysis performed using GraphPad Prism 8.0.I2 = 98.61%, p <. 001). The prevalence was higher in the pre-ART era compared to the ART era, but not to statistical significance . In those diagnosed by endoscopy, the prevalence was higher compared to patients diagnosed by non-endoscopic approaches, but not to statistical significance . The prevalence of OC significantly decreased over the study period .Thirteen eligible studies from 9 SSA countries including a total of 113,272 patients were qualitatively synthesized, and 9 studies were included in the meta-analysis. Overall pooled prevalence of HIV-associated OC was 12% : 8 to 15%, The prevalence of OC among PLHIV in the ART era in SSA is decreasing. However, OC remains a common problem. Active endoscopic surveillance of symptomatic patients and further empirical studies into the microbiology, optimal antifungal treatment, and impact of OC on quality of life of PLHIV in SSA are recommended. Sub-Saharan Africa (SSA) remains the region most affected by the human immune deficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) pandemic . Of the 3 [Mucocutaneous manifestations due to opportunistic infections and malignancies are the most common and usually the first recognizable clinical presentation in patients with HIV/AIDS , 4. Pers3 . OC typi3 . High HI3 .Candida albicans but also other non-albicans Candida species such as C. glabrata, C. tropicalis, and C. dubliniensis [Candida species and is the preferred method of diagnosis of OC [OC is the most common cause of infectious esophagitis in patients with advanced HIV disease, mainly caused by the yeast iniensis . OC is ainiensis . On endoiniensis . Biopsy is of OC . MicrobiBetween 10 and 15% of PLHIV will develop esophageal candidiasis at one point in their life-time . Early iWe hypothesized that with over 10 years of the widespread use of ART and enhanced anti-infective prophylaxis in SSA, the occurrence of opportunistic diseases including OC is expected to have significantly reduced. However, there is a scarcity of studies to demonstrate the trend of OC in the pre-ART and ART era in SSA. Such information is not only valuable for epidemiological purposes but also help guide targeted clinical care of this vulnerable population. The aim of this study, therefore, was to determine the prevalence and the trend in the prevalence of OC among PLHIV in SSA over the past nearly 40 years of the HIV/AIDS pandemic.This systematic review and meta-analysis were conducted according to the criteria outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . The PRIWith the help of a qualified medical librarian, all studies published from January 1980 to June 2020 were searched from EMBASE, PubMed, Web of Science, and the African Journals Online (AJOL).Candida infection of the oesophagus” OR “monilial oesophagitis” AND “human immune deficiency virus” OR “HIV” OR “acquired immune deficiency syndrome” OR “AIDS” AND “Africa South of Sahara” OR “sub-Saharan Africa” OR “Angola” OR “Benin” OR “Botswana” OR “Burkina Faso” OR “Burundi” OR “Cameroon” OR “Cape Verde” OR “Central African Republic” OR “Chad” OR “Comoros” OR “Democratic Republic of Congo” OR “Cote d'Ivoire” OR “Eritrea” OR “Eswatini” OR “Ethiopia” OR “Gabon” OR “Gambia” OR “Ghana” OR “Guinea” OR “Guinea-Bissau” OR “Kenya” OR “Lesotho” OR “Liberia” OR “Madagascar” OR “Malawi” OR “Mali” OR “Mauritania” OR “Mauritius” OR “Mozambique” OR “Namibia” OR “Niger” OR “Nigeria” OR “Rwanda” OR “Sao Tome and Principe” OR “Senegal” OR “Seychelles” OR “Sierra Leone” OR “Somalia” OR “South Africa” OR “South Sudan” OR “Sudan” OR “Tanzania” OR “Togo” OR “Uganda” OR “Zaire” OR “Zambia” OR “Zimbabwe”.The following search terms were used: “oesophageal candidiasis” OR “esophageal candidiasis” OR “candidal oesophagitis” OR “candidal esophagitis” OR “Besides, we performed a manual literature search for all the references of the included articles to find applicable studies. The search was limited to human studies only. Studies written in English and French, which are the most common languages, published in sub-Saharan Africa were selected.Duplicate studies were identified and merged using the HDAS program. Titles and abstracts were primarily screened by two independent reviewers (F.B and R.O) to exclude those not related to this study. After the initial screening, full texts of potentially eligible studies were retrieved and examined for eligibility.i.Retrospective or prospective observational studies from January 1980 to June 2020.ii.Published in the English and French languages.iii.Reporting prevalence of HIV-associated OC in countries located in SSA.Studies that met the following inclusion criteria were included:Studies reporting oropharyngeal candidiasis or did not specify the location of upper gastrointestinal candidiasis were excluded. Single-case reports and case series reporting less than 10 cases were also excluded from the study.Study characteristics; authors, year of publication, study country, study design.Study characteristics; age (mean or median), sex, the sample size of the participants, number of patients diagnosed with OC , how the diagnosis was made (non-endoscopic vs. endoscopy), and the period of data collection in each study.A pre-designed macros form in Microsoft Excel 2016 was used to extract data from each article. The following items were extracted:Two independent reviewers (RO and FB) extracted data, and any differences were resolved by discussion and consensus. The corresponding authors for eligible studies with missing data were contacted by email for clarification.The pre-ART era was defined as patients diagnosed with HIV-associated OC before 2004 (1981–2003) and ART era between 2004 and to date. ART era was further sub-divided into early ART era (2004–2014) when ART was limited to only severely ill patients   and lateAll the eligible studies included were assessed for risk of bias using the Critical Appraisal Skills Programme (CASP) checklist. The risk of bias in individual studies was graded as low, moderate, and high. Studies with a high risk of bias were excluded from the meta-analysis.The study outcome is the pooled prevalence and the trend in the prevalence of OC in patients with HIV/AIDS living in SSA.I2 value. A funnel plot was generated to assess for publication and sensitivity analysis performed. Mann-Whitney test was performed in GraphPad Prism 8.0. p < 0.05 was considered statistically significant at the 95% confidence interval (CI). Descriptive statistics were used to summarize data from individual studies. A narrative synthesis was also employed to present the results and discussion of the different studies included.All analyses were performed using Microsoft Office 2016 , GraphPad Prism 8.0, and STATA 16.0 statistical software . A random effect cumulative meta-analysis was performed using in STATA 16.0 to illustrate the trends in the prevalence of HIV-associated OC and presented in a forest plot. Heterogeneity across studies was assessed using Q statistics and n = 1; Togo, n = 2; Cameroon, n = 1; Malawi, n = 2; Mozambique, n = 1; Zambia, n = 1; Nigeria, n = 1; Kenya, n = 1; and Uganda, n = 3;) were eligible from the studies identified through database and manual literature search  were retrospective studies, and one was a randomized clinical trial. Five of the studies were conducted before the introduction of ART.Table I2 = 98.61%, P < 0.001), Fig. Candida species responsible.Four of the 13 studies recruited a more restricted group of patients –28, withOverall, the prevalence of OC significantly decreased over the over three decades of the HIV pandemic in SSA, Figs. p = 0. 071), Fig. In 7 studies, diagnosis of OC was solely made through upper gastrointestinal endoscopy, non-endoscopic  in 5 studies, and not specified in one study Table . Overallp = 0.095, Fig. p = 0.071), Fig. Five studies were conducted during the pre-ART era and 8 studies during the ART era Table . The meaThis systematic review aimed to determine the prevalence and the trend in the prevalence of OC in the pre-ART and ART era. Overall, in this large review, the pooled prevalence of OC was 12% , which is comparable to the global data on the lifetime prevalence (10 to 15%) of OC among patients with HIV/AIDS . A signiIn a large study evaluating over 100,000 PLHIV in Uganda, the prevalence of OC was stratified into pre-ART period , early ART  and late ART  . Howeverp < .05) with up to 8.8% of deaths reported [3, and only 23.1% of the patients in the study were on ART. Mohamed and colleagues found that more esophageal lesions were significantly associated with CD4 counts less than 200 cells/mm3 [3 in a Ugandan prospective study and the median survival time was 3.1 months in the untreated AIDS patients [In Malawi, OC significantly correlated with inpatient mortality  over several days of antifungal therapy is considered an alternative diagnostic approach . FluconaC. albicans, the most common cause of OC is usually susceptible to commonly prescribed triazole antifungals (fluconazole) [albicans Candida species, treatment failure is common. C. krusei is intrinsically resistant to fluconazole but C. dubliniensis and C. glabrata which are more common in HIV have high minimum inhibitory concentrations to this commonly used antifungal [Lack of response to systemic antifungal response is uncommon and usually due to antifungal resistance or a wrong clinical diagnosis . C. albionazole) . Howevertifungal . Therefotifungal . In thostifungal .This study has some important limitations. Firstly, in over 10,000 cases of OC, the method of diagnosis was not specified or the disease was diagnosed clinically. This increases the risk of overestimation of the true burden of the disease; especially that HSV esophagitis and other viral esophagitis are equally common in the African population. Secondly, microbiological confirmation and speciation of the etiologic confirmation were not reported in all studies. These are very important areas for future studies. Thirdly, there was a very high degree of statistical heterogeneity in this study. This is due to the vast difference in the diagnostic methods, the population studied (denominator), and study design. However, we minimize this by performing sensitivity analysis and constructing a cumulative prevalence analysis in addition to the traditional pooled meta-analysis.Overall, our pooled data from this systematic review suggest a trend towards a declining prevalence of OC among PLHIV in the ART era in SSA. However, OC remains a common problem among PLHIV. Treatment outcomes and etiology of the disease largely remain unknown. This has a clinical implication in the choice of empirical antifungal therapy for suspected cases. Continuous active surveillance through OGD, empirical studies into microbiology and optimal antifungal treatment, and the impact of OC on quality of life of PLHIV in SSA are subjects for future studies.


Compared to SAT, adipocytes in PPAT were smaller, had the same basal rates of fatty acid release (lipolysis) yet released less polyunsaturated fatty acid species, and were more sensitive to isoproterenol-stimulated lipolysis. Basal lipolysis of PPAT was increased in men diagnosed with less aggressive PCa (Gleason score (GS) ≤ 3 + 4) compared to men with more aggressive PCa (GS ≥ 4 + 3) but no other measured adipocyte parameters related to PCa aggressiveness. Likewise, there was no difference in PPAT lipid biology between lean and obese men. In conclusion, lipid biological features of PPAT do differ from SAT; however, we did not observe any meaningful difference in ex vivo PPAT biology that is associated with PCa aggressiveness or obesity. As such, our findings do not support a relationship between altered PCa behavior in obese men and the metabolic reprogramming of PPAT.The prostate is surrounded by periprostatic adipose tissue (PPAT), the thickness of which has been associated with more aggressive prostate cancer (PCa). There are limited data regarding the functional characteristics of PPAT, how it compares to subcutaneous adipose tissue (SAT), and whether in a setting of localized PCa, these traits are altered by obesity or disease aggressiveness. PPAT and SAT were collected from 60 men (age: 42–78 years, BMI: 21.3–35.6 kg/m Prostate cancer (PCa) is the most commonly diagnosed cancer in men in developed countries and the second most common cancer worldwide . SeveralSignificant attention has focused on the role that the local adipose bed that surrounds the prostate, called periprostatic adipose tissue (PPAT), plays in influencing PCa biology. For example, higher grade or more aggressive PCa has been linked to greater PPAT thickness , adiposIn this study, we assess the lipolytic profile and adipocyte size of PPAT and compare it to that of SAT in men undergoing radical prostatectomy for localized PCa. Further, we explore the relationship between adipose lipid biology and metabolic profiles and PCa clinical features. We hypothesize that lipid-attributes of PPAT differ from SAT, and that PPAT lipolysis is increased in men with more aggressive PCa and in obese men.2 ± 3.4 (range: 21.3 to 35.6), indicating a slightly overweight population. The mean waist circumference (WC) was 95.8 cm ± 8.7 (range: 81 to 117), indicative of a WC linked with an elevated risk of all-cause mortality [The patient cohort was comprised of 60 patients with a mean age of 62 years ± 8  and LDL  in patients taking statins. Blood serum glucose was significantly elevated in statin patients .Of the 60 patients assessed, 9 patients were using statin medication. There was no difference in the average age, BMI, and WC between the men taking statins compared to men not taking statins, as expected. There was the expected reduction in circulating cholesterol .It is well established that there are anatomical, cellular, molecular, and physiological differences between SAT and visceral adipose tissue of the abdominal cavity . However2 = 0.004, p = 0.6; SAT: R2 = 0.0009, p = 0.8), or with mean adipocyte size .The rate of basal fatty acid release from PPAT and SAT explants was similar C; howeveNext, we determined the abundance of fatty acid species that were released by PPAT and SAT in the basal state. Of the 28 species measured, 6 species were less abundant in the secretome of PPAT, specifically 15:1 n-10, 18:3 n-6, 20:3 n-6, 20:4 n-6, 22:5 n-3, and 22:6 n-3, and total polyunsaturated fatty acids (PUFAs), both n-6 and n-3 PUFAs, were less abundant compared to SAT . Conversp = 0.0009; We next assessed the responsiveness of PPAT and SAT explant lipolysis to hormone stimulation. As expected, insulin suppressed lipolysis, whereas both concentrations of isoproterenol increased lipolysis in both PPAT and SAT explants . Howeverp = 0.054), whilst PSA and age were greater in the “≥4 + 3” cohort compared to the “≤3 + 4” cohort (A Gleason score (GS) is used to grade clinical PCa pathology. We dichotomized our cohort into a less aggressive “GS ≤ 3 + 4” and a more aggressive group “GS ≥ 4 + 3” since patients with “≥4 + 3” disease have greater rates of relapse compared to “≤3 + 4” . From th” cohort .There was no difference in the mean size or the distribution of adipocyte sizes between GSs of “≤3 + 4” (less aggressive) and “≥4 + 3”  in men with high WC compared to men with low WC, with LDL (p = 0.07) trends being elevated in patients with a high WC (p = 0.13). PSA (p = 0.74) and age (p = 0.46) were not different between patients with normal or high WC, as expected (Waist circumference (WC) is a better predictor of all caused mortality than BMI in populations ≥50 years old ,33, and  high WC . There wexpected .Mean PPAT adipocyte size, and the distribution of adipocyte sizes, was not different between low and high WC patients A,B. Likep = 0.13; p = 0.40 main effect by ANOVA), nor between low and high WC . Finally, we saw similar patterns in SAT . To In this study, we observed lower levels of PUFAs  being secreted from PPAT explants compared with those secreted from SAT. The reduction in the levels of secreted fatty acid species may be simply due to less of those species being stored within adipocytes of adipose tissue. To date, intracellular PPAT and SAT fatty acid species has not been defined. That said, one study did report that the levels of PUFA 20:4 n-6 extracted from PPAT  obtained from men with PCa were lower than from the control group (benign prostatic hyperplasia) . We did Several studies have shown that PPAT-derived signals, including proteins and lipids, can exacerbate PCa progression in vitro ,48,61. Cp = 0.054), but there was no difference in mean BMI. Whilst we did not see changes in PPAT lipid biology in lean or obese men, this does not negate the role of other aspects of PPAT biology that may influence PCa aggressiveness. For example, the gene expression profile of PPAT from obese men favors hypercellularity and reduced immune-surveillance [Like many other cancer types , PCaeillance , as welleillance  and increillance . PPAT freillance . Likewiseillance . Importaeillance . It is ieillance  and so tAnterior PPAT and abdominal SAT were removed and collected from men that were diagnosed with localized PCa, who opted for radical prostatectomy via a robotic da Vinci technique at St Vincent’s Hospital. Clinical data of the patients, prostate, and PCa was obtained from the APCRC-NSW/Australian Prostate Cancer BioResource. Patient data, including anthropometric data, current medication, and familial history, were collected at St Vincent’s Hospital prior to radical prostatectomy and collated by the APCRC-NSW/Australian Prostate Cancer BioResource. PCa pathological analysis and prostate characterization were conducted at Douglass Hanly Moir Pathology . Histopathological data, including lymph node invasion, extracapsular extension, and PCa grading via ISUP and Gleason score , were provided to and collated by the Australian Prostate Cancer BioResource. Analysis of patient blood serum was undertaken by Royal Prince Alfred Hospital Pathology laboratory, Sydney, NSW, Australia.The study was approved by the St. Vincent’s Hospital Sydney Research Ethics Committee (12/231).2, 1.3 mM KH2PO4, 1.3 mM MgSO4·7H2O, 24.8 mM NaHCO3), 2% fatty acid-free BSA, and 5.5 mM glucose. Explants were treated with either 10 nM insulin , 1 μM  and 10 nM (mid-dose) dl-Isoproterenol (Sigma-Aldrich), and 1 nM dihydrotestosterone  or dH2O (control) for 2 h in a 37 °C water bath. Media non-esterified fatty acids levels was determined using commercial kit following manufacturer’s instructions . Media fatty acids were determined using gas chromatography, as described below.PPAT and SAT explants were dissected into five pieces (~30–50 mg each) and incubated in Kreb’s buffer . Images of three randomized fields were collected, with each field containing a minimum of 60 cells, as previously defined : “minimup ≤ 0.05 considered significant.GraphPad Prism version 8.4.1 for PC  and Microsoft Excel  were used to statistically analyze all data, with For dichotomized analysis , non-parametric Wilcoxon signed-rank and Mann–Whitney U-tests were used to determine significance. Linear regression models were used to test for relationships between dichotomized groups. Non-parametric Kruskal–Wallis ANOVAs were used to test for main effect, while posthoc Tukey’s multiple comparisons and Dunn’s multiple comparisons were undertaken. Two-way ANOVAs were conducted to test for main effect, while posthoc Tukey’s multiple comparisons were undertaken.The data presented show that the lipid biological features of PPAT do differ from SAT; however, we did not observe any meaningful difference in ex vivo PPAT biology that align with differences in PCa aggressiveness or patient metabolic health. Significantly, this is the first study to comprehensively assess PPAT lipid biology and compare it to paired SAT. In combination with other studies that reported that PPAT thickness ,43, adip


N-acetylgalactosaminyltransferase (GALNT) family function as the initiating enzymes that catalyze mucin-type O-glycosylation of proteins, and their dysregulated expression can alter cancer cell behaviors such as de novo occurrence, proliferation, migration, metastasis, and drug resistance. Recent studies have demonstrated that one of the family’s members, GALNT14, is aberrantly expressed in multiple cancers and involved in a variety of biological functions. Moreover, the single nucleotide polymorphisms (SNPs) of GALNT14-rs9679162 have been shown to predict therapeutic outcomes in patients with hepatocellular carcinoma as well as several other different types of gastrointestinal cancer. This review summarizes the structural features of GANLT14, its functional roles, and the predictive values of GALNT14 genotypes and enzyme levels in multiple cancers receiving distinct anticancer therapies.Members of the polypeptide  N-acetylgalactosaminyltransferase (GALNT) family are enzymes that initiate O-glycosylation by the addition of an N-acetylgalactosamine  to a serine or threonine residue of a mucin-type protein [Glycosylation, an enzymatic process that attaches glycans to proteins or other organic molecules, is essential for multicellular life. Among several types of glycosylation, the addition of a sugar to the oxygen atom of amino acid residues (O-glycosylation) occurs on thousands of secreted and cell surface proteins, altering their structures, functionalities, and subcellular distributions ,2,3,4. O protein . This pr protein . The GAL protein . In canc protein . GALNT14 was firstly cloned and identified from the gastric cancer cell line MKN45 in 2003 [GALNT14 gene is located at chromosome 2p23.1, spanning over 228 kb and containing at least 17 exons , as assessed using the kernel-based association test. Secondly, validation of SNP rs9679162 in association with therapeutic responses was performed in an independent cohort of 41 patients prospectively. In this validation cohort, a significant association was found again between rs9679162 and the therapeutic responses (p = 0.006326). A follow-up survival analysis demonstrated that patients with the “TT” genotype had better progressive-free survival (PFS) in both retrospective and prospective cohorts  than “non-TT” (“GT” + “GG”) genotypes. However, the overall survival (OS) was only significantly different in the retrospective cohort (p = 0.00622), not in the prospective cohort. This study demonstrated firstly that GALNT14-rs9679162 genotypes are potentially associated with the responses of the first course of FMP chemotherapy in patients with advanced HCC and could serve as a predictor marker for HCC. The predictive value of the GALNT14-rs9679162 SNP in cancer was first discovered in a study attempting to identify germline SNP markers capable of predicting the treatment responses of advanced HCC patients receiving chemotherapy using 5-fluorouracil, mitoxantrone, and cisplatin (FMP) . Two expp < 0.001) and PFS (3.9 vs. 2.1 months p < 0.001), and a better response rate  and disease control rate . A further multivariate analysis confirmed that the rs9679162 genotype was an independent predictor for OS (p = 0.002). A categorical analysis showed that a subgroup of patients with the “TT” genotype, who had a tumor size <10 cm and neutrophils <74%, had the best outcome . This prospective study confirmed that the GALNT14-rs9679162 SNP is an effective predictor for therapeutic outcomes in advanced HCC patients receiving split-dose FMP chemotherapy.To confirm the predictive value of GALNT14-rs9679162, a prospective study was conducted in advanced HCC patients receiving a less toxic, split-dose FMP protocol . One hunp = 0.001, 0.035, 0.008, and 0.009, respectively) and OS . A total of 30 patients (25.4%) with both AFP ≤2800 ng/mL and the GLANT14 “TT” genotype exhibited longer median PFS and OS , and nine patients (7.6%) with all four favorable factors exhibited the longest median PFS and OS . These results suggest that a lower AFP level in combination with the GALNT14 “TT” genotype could serve as favorable pre-therapeutic predictors for advanced HCC patients receiving FMP chemotherapy.To evaluate whether a combination of the SNP and other clinical parameters together with on-treatment side effects could serve as an effective predictor for favorable outcomes, 118 advanced HCC patients receiving split-dose FMP were retrospectively enrolled in the study . Resultsp = 0.023). The proportion of the “TT” genotype in Chinese population was shown to range from 24.18% to 30.15% in different cohorts. It was significantly higher in Japanese and African populations , but significantly lower in an Italian cohort . The distribution of different GALNT14 genotypes in HCC has been demonstrated to be related to HCC etiologies and ethnicities . The “TTp < 0.001) and time-to-complete response  and a longer PFS (p < 0.001), when compared with the “non-TT” genotype. In patients with albumin <3.5 g/dL, the “TT” genotype was associated with longer OS (p = 0.027). This study showed that GALNT14 genotypes were also significantly associated with clinical outcomes of HCC patients receiving TACE.TACE is currently the standard treatment in HCC patients with Barcelona Clinic Liver Cancer stage B. A cohort of 327 HCC patients treated with TACE was enrolled to investigate the predictive prognosis value of the GALNT14-rs9679162 genotype . Again, p = 0.012 and 0.002). Moreover, the WWOX-rs13338697 “CT” genotype was associated with an unfavorable TTP (p = 0.031), and the rs6025211 “CT” genotype was associated with an unfavorable OS (p = 0.014). When these three SNPs were combined, patients with GALNT14-rs9679162 “TT”/WWOX-rs13338697 “non-CT” genotypes achieved the most favorable treatment outcomes , respectively), whereas patients with GALNT14-rs9679162 “non- TT”/rs6025211 “CT” genotypes were associated with the most unfavorable treatment outcomes , respectively). The remaining patients had intermediate clinical outcomes. This study suggests that pretreatment genotyping of these SNPs could help to decide whether these patients should receive chemotherapy or other treatments.GALNT14-rs9679162 has been demonstrated to be capable of predicting chemotherapy responses in advanced HCC . Howeverp = 0.024), compared with the remaining patients. When the three favorable SNP markers were combined with two clinical criteria , the complete response rate to chemotherapy reached 60%. Subsequent sorafenib for chemotherapy non-responders was associated with longer OS (p < 0.01). Surprisingly, the GALNT14 “GG” genotype was associated with longer OS  in HAIC treated patients.Although targeted agents are recommended as the first-line treatment for advanced HCC, they were not able to significantly prolong survival in certain advanced HCC patients with distal metastasis. On the other hand, systemic chemotherapy or hepatic arterial infusion chemotherapy (HAIC) could achieve a complete response in a small proportion of patients. In order to identify the subgroups of HCC patients with the best outcomes out of those receiving either chemotherapy, HAIC, or targeted agents, previously identified SNP predictors  were tested in a real-world cohort of 237 advanced HCC patients  . The resp = 0.027) and the “GG” genotype was associated with a shorter OS (p = 0.006) in patients who were positive for the anti-hepatitis C virus antibody. In patients with who were positive for the hepatitis B virus surface antigen, no significant association was found between the genotypes and prognosis . To evaluate the predictive role of GALNT14-rs9679162 in patients receiving sorafenib treatment, a cohort of 81 HCC patients treated by sorafenib as the first-line therapy was studied . The resGALNT14-rs9679162 SNP has been studied in various gastrointestinal (GI) cancers, including esophageal squamous cell carcinoma, gastric signet ring cell carcinoma, colorectal adenocarcinoma, pancreatic adenocarcinoma, and cholangiocarcinoma . Further multivariate Cox-proportional hazard model analysis showed that the “GG” genotype was associated with a longer time to complete/partial response . Since the presence of a complete/partial response to CCRT was critical for advanced ESCC patients to achieve better outcomes, the “GG” genotype can be used as an unfavorable predictor for CCRT in advanced CCRT. Concurrent chemoradiotherapy (CCRT) is the most common treatment for patients with locally advanced, unresected esophageal squamous cell carcinoma (ESCC) to prolong patient survival ,26. Howep = 0.048) in advanced stage SRCC patients (TNM stage IIB to IV). The subgroup analysis further revealed that the ”TT” genotype was associated with unfavorable OS in SRCC patients harboring more aggressive phenotypes such as node status > 0 , lymphatic invasion , vascular invasion , and perineural invasion . This suggested that gastric SRCC could be stratified into different prognostic subgroups by the combination of clinicopathological factors and the GALNT14 genotype. The poorest prognosis subgroup included patients with the aggressive phenotype together with the GALNT14 “TT” genotype.Gastric signet ring cell carcinoma (SRCC) is one type of gastric cancer carrying a poorer prognosis compared with other types of gastric cancer . Howeverp = 0.019). A subgroup analysis further showed that the “TT” genotype was associated with an unfavorable OS in the following subgroups: age ≤ 65 years, male, left side CRC, N2 stage, carcinoembryonic antigen >5 ng/mL, and mucinous histology . This study concluded that the “TT” genotype is an unfavorable prognostic marker in patients with stage III colorectal adenocarcinoma receiving oxaliplatin-based adjuvant chemotherapy. Adjuvant oxaliplatin-based chemotherapy has been suggested as a standard treatment for patients with stage III colorectal adenocarcinoma following surgical resection. Although adjuvant chemotherapy has been shown to reduce around 30% of disease recurrence and 22% to 32% of mortality, not all patients benefit from adjuvant therapy ,32. Therp = 0.005). Further univariate analysis followed by multivariate Cox proportional hazard analysis identified the “GG” genotype, negative resection margin, and locoregional disease as independent predictors for favorable OS . The study suggested that the “GG” genotype could serve as a favorable prognostic marker in patients with resected PDA.Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancers and is associated with a poor prognosis due to its advanced presentation at diagnosis and limited therapeutic options. The lack of validated predictive markers further complicates this situation . Again, p = 0.023). Furthermore, two tumor characteristics, perineural and vascular invasion, were independently associated with unfavorable OS . In a multivariate linear analysis, the “TT” genotypes were independently associated with two known predictors of unfavorable prognosis, perineural invasion (p = 0.035) and lymph node metastasis (p = 0.005). This study concluded that the “TT” genotype was associated with perineural invasion and lymph node metastasis, as well as unfavorable OS in patients with resected cholangiocarcinoma.Cholangiocarcinoma emerges from the dysregulated proliferation of cholangiocytes and is notorious for its poor prognosis and response to chemotherapy . The assGALNT14-rs9679162 SNP showed that in HCC and esophageal cancer (“carcinoma”), patients with the “TT” genotype have a better prognosis, whereas in other cancers (“adenocarcinoma”), patients with the “GG” genotype have better outcomes [GALNT14 genotype were invested in a cohort of 44 patients who had undergone surgical resection of HCC [p = 0.008 and < 0.001 for “TT” and “non-TT” genotypes, respectively), and (ii) a significantly higher cancer to noncancer (C/non-C) ratio of GALNT14 expression was noted in the “TT” genotype compared with the “non- TT” genotype (p = 0.001). Additionally, the C/non-C ratios of cFLIP-S were significantly lower in the “TT” genotype than those in the “non-TT” genotype (p = 0.014). Glycoproteomic analysis showed that the glycosylated residues of death receptor 5 (DR5) in the “TT” patient-derived HCC tissues were clustered in the two major glycosylation sites, in contrast to a scattered glycosylation pattern in the “non-TT” liver tumor tissues. These molecular findings suggest that the cancerous tissues from HCC patients with the “TT” genotype are more sensitive to extrinsic apoptosis signaling compared with those with the “non-TT” genotype. Previous studies of the predictive role of the outcomes ,33,35,37outcomes  and PDA n of HCC . AnalysiIn PDA, a preliminary assessment of the GALNT14 enzyme expressions from 20 patients (10 with “TT” and 10 with “GG” genotype) was performed . Among tp = 0.0015). GALNT14 promotes breast cancer metastasis to the lungs by enhancing the initiation of metastatic colonies and their subsequent growth into overt metastases. The inhibitory effect of lung-derived bone morphogenetic proteins (BMPs) on cancer self-renewal was overcome by GALNT14, which facilitated metastasis initiation within the lung microenvironment. In addition, GALNT14 not only enhanced the recruitment of macrophages to the site of metastases, but also exploited macrophage-derived fibroblast growth factors (FGFs). Moreover, KRAS-PI3K-c-JUN signaling was identified as an upstream pathway that accounted for the elevated expression of GALNT14 in lung-metastatic breast cancer. In a study conducted with the MCF-7 breast cancer cell line, overexpression of GALNT14 significantly enhanced cell proliferation, migration, and tumor invasion, while the knockdown of GALNT14 reduced clonogenicity and attenuated cell migration and cell invasion [GALNT14 [p = 0.038), higher clinical stages (p < 0.0001), and shorter DFS (p = 0.029). Another study showed that GALNT14 regulated the stability of P-pg, the efflux pump localized on the cell membrane, which further promoted multidrug resistance [The association between cancer characteristics and GALNT14 protein expression has been studied in breast cancer . GALNT14invasion . Moreoveinvasion . The che[GALNT14 . Furthersistance . These sGALNT14 significantly suppressed cell migration and cellular morphology change through aberrant glycosylation of transmembrane mucin 13 [GALNT14 as a direct target of miR-125a, and overexpression of miR-125a markedly reduced the expression of GALNT14. Both miR-125a mimics and GALNT14 siRNA suppressed the activity of matrix metalloproteinase (MMP)-2 and MMP-9, further inhibiting extracellular matrix degradation. A recent study showed that aberrant expression of BORIS (Brother of the Regulator of Imprinted Sites) altered cell migration and invasion via upregulation of GALNT14, suggesting that BORIS is a potential therapeutic target in ovarian high-grade serous carcinoma [Overexpression of GALNT14 was found in ovarian cancer . Knockdomucin 13 . Moreovearcinoma .GALNT14 increased cellular Apo2L/TRAIL sensitivity, whereas RNA interference of GALNT14 expression reduced responsiveness. Through the modulation of extracellular DR4 and DR5 O-glycosylation sites, GALNT14 enhanced apoptotic signaling. Immunohistochemistry assays that measured GALNT14 expression have been developed to select NSCLC patients who might be more sensitive to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) [The expression of GALNT14 was highly associated with a shorter RFS in a clinicogenomics study that included 138 NSCLC patients . In PDA,ntibody) . Moreoventibody) . A meta-GALNT14 mutation (c.802C > T) was identified as a neuroblastoma predisposition gene and was predicted to be functionally damaging by the PolyPhen2 (Polymorphism Phenotyping v2) and SIFT (Sorting Intolerant From Tolerant) scoring methods [GALNT14 is located close to ALK on 2p23.1, a region previously discovered to be linked with neuroblastoma.A  methods . FurtherGALNT14 as well as its SNP genotype can be used to predict clinical outcomes in various types of cancer. However, the relationships between the GALNT14 SNPs, the enzyme expressions, and the tumor behaviors have not been fully unveiled. Here, we summarized the current knowledge of GALNT14 in various cancers, particularly focusing on its role as a biomarker. Further studies are still needed to provide the missing mechanisms regarding the links between the genotypes and expression levels, as well as other GALNT14-involved microenvironmental elements that can modulate cancer behaviors. For example, HCC is tightly linked to liver cirrhosis and male gender, and thus it will be interested to know whether GALNT14 is involved in modulating fibroblast or sex hormone behavior. Hopefully, with this knowledge, new therapeutic strategies can be devised in the future.GALN14, an initiating enzyme of O-glycosylation, has been demonstrated to play pivotal roles in cancer cell proliferation, migration, and metastases. The expression level of


Dementia with Lewy bodies (DLB) is the second most prevalent neurodegenerative dementia after Alzheimer’s disease, and is pathologically characterized by formation of intracellular inclusions called Lewy bodies, the major constituent of which is aggregated α-synuclein (αS). Currently, neither a mechanistic etiology nor an effective disease-modifying therapy for DLB has been established. Although two missense mutations of β-synuclein (βS), V70M and P123H, were identified in sporadic and familial DLB, respectively, the precise mechanisms through which βS mutations promote DLB pathogenesis remain elusive. To further clarify such mechanisms, we investigated transgenic (Tg) mice expressing P123H βS, which develop progressive neurodegeneration in the form of axonal swelling and non-motor behaviors, such as memory dysfunction and depression, which are more prominent than motor deficits. Furthermore, cross-breeding of P123H βS Tg mice with αS Tg mice worsened the neurodegenerative phenotype presumably through the pathological cross-seeding of P123H βS with αS. Collectively, we predict that βS misfolding due to gene mutations might be pathogenic. In this paper, we will discuss the possible involvement of amyloidogenic evolvability in the pathogenesis of DLB based on our previous papers regarding the P123H βS Tg mice. Given that stimulation of αS evolvability by P123H βS may underlie neuropathology in our mouse model, more radical disease-modifying therapy might be derived from the evolvability mechanism. Additionally, provided that altered βS were involved in the pathogenesis of sporadic DLB, the P123H βS Tg mice could be used for investigating the mechanism and therapy of DLB. Dementia with Lewy bodies (DLB) is the second most prevalent neurodegenerative dementia after Alzheimer’s disease (AD) . A histoMissense mutations of disease-relevant amyloidogenic proteins (APs) can be rarely associated with clinical neurodegeneration in humans. For instance, in DLB, one missense mutation of α-synuclein (αS), E46K, was identified in familial DLB , while tRecently, we proposed that neurodegeneration in aging may be attributed to amyloidogeioc evolvability ,12,13. IAt present, numerous PD and other α-synucleinopathy models exist where αS cDNA is overexpressed under various promoters . Yet, asIn our study, Tg mice were created using Thy-1 promoter to drive familial DLB-derived P123HβS cDNA a 14]. T. T14]. TSimilar to human DLB patients, P123H βS Tg mice showed behavioral abnormalities, with the non-motor symptoms being more prominent than motor deficits  Figure . WhereasTo investigate the combined effect of P123H βS and αS, P123H βS Tg mice were subjected to cross-breeding with αS Tg mice ,21. The Collectively, many behavioral abnormalities in both single P123H Tg mice and bigenic mice manifested earlier compared to formation of inclusion bodies. These results may suggest that small aggregates, including oligomers and protofibrils of APs, rather than mature fibrils of APs, might be attributed to behavioral alterations. Alternatively, protein aggregation of APs might be not related to alteration of behaviors in neurodegeneration.DLB has overlapping features of both AD and PD  and mighMore recently, Tg mice expressing familial DLB-linked E46K αS were also reported . AlthougIt is expected that a better understanding of the pathogenic mechanism of neurodegeneration may lead to development of evidence-based therapy for neurodegenerative diseases, including DLB. Although clinical trials have been extensively performed based on several hypotheses for the mechanism of neurodegeneration, the results are so far unsatisfactory.Currently, most prevailing views suggest that neurodegeneration may be attributed to the accumulation of the neurotoxic aggregates of amyloidgenic proteins. In this context, “amyloid cascade hypothesis” (ACH), a dominant hypothesis in AD, postulates that aggregation of Aβ triggers a toxic cascade of events, including aberrant phosphorylation of tau, neuritic plaque and neurofibrillary tangle formation, synapse loss, and neuroinflammation, eventually leading to neuronal death . AccordiIn the meantime, much attention had been paid to the role of other factors, such as inflammation and oxidative stress, in the pathogenesis of aging-associated neurodegenerative diseases. In support of the former, several epidemiological studies have shown that nonsteroidal anti-inflammatory drugs may reduce the risk of development of AD and PD. However, clinical studies of ibuprofen and other structurally related compounds for AD have been unsuccessful ,30. In tOne may therefore consider that a better understanding of the physiological functions of APs is necessary for successful therapy development in neurodegenerative diseases. In this context, it is noteworthy that Aβ was rapidly seeded by herpes simplex virus (HSV) 1 to protect against brain infection in cellular and Tg mice models of AD, suggesting that fibrillation of Aβ might play a protective role in innate immunity of the nervous system . ConsistSince elucidation of both pathological and physiological functions of APs might be critical for developing effective therapy for neurodegenerative diseases, we recently proposed that evolvability is a physiological function of APs. The concept of evolvability is based on the evolvability of yeast prion that is critical for yeast surviving in the stressful environment . FurtherAs with related conditions, we have posed the question as to why such neurodegenerative conditions as DLB might not only appear but persist across evolutionary pressures. In this regard, our recent view regarding amyloidogenic evolvability might provide a clue to address this question. Regarding the physiological function of APs relevant to neurodegeneration including αS, we recently proposed that amyloidogenic evolvability might be important in the brain exposed to multiple stressors, including hyperthermia, oxidative stress, and neurotoxicity . PreciseConsidering the missense mutations in αS, following discovery of an A53T substitution in PD , two othRecently, several genome wide association studies (GWAS) have linked various genes to DLB. Curiously, the glucocerebrocidase (GBA) gene, which encodes a lysosomal enzyme involved in sphingolipid degradation, is deleted in Gaucher disease. It is conceivable that the linkage of Gaucher disease to sporadic PD  may implFurthermore, it is intriguing to hypothesize that alterations of wild type βS in aging, presumably through an antagonistic pleiotropy mechanism, might play a central role in DLB pathogenesis . As βS mThen, by what mechanism is dementia, the major symptom in DLB, manifested? Given the association of βS with APs, including Aβ and αS, two possibilities exist. Notably, synelfin, the avian form of αS, is essential for bird song memory formation during a critical period in development . If αS iAlternatively, cognitive deficits characterized in the P123H βS Tg mice might be derived from aggregation of endogenous AD-relevant APs, including Aβ and tau, which might be increased by expression of P123H βS. Thus, further studies are warranted to investigate such endogenous APs in P123H βS Tg mice. If cross-breeding of the P123H βS Tg mice with AD-related mice, such as either APP Tg mice or APP knockout mice, is possible, this might provide insight into such interactions. As described above, wild type βS might be altered in sporadic DLB through antagonistic pleiotropy, thus promoting AP aggregation. If correct, then familial and sporadic DLB may share essentially similar mechanisms underlying dementia. Therefore, P123H βS Tg mice might be applicable for the study of all types of DLB.At present, no disease-modifying therapies can either arrest or slow neuronal damage in DLB. As such, current strategies focus mainly on symptomatic treatments, such as cholinesterase inhibitors, which modestly improve cognition in AD and are also approved for Lewy body dementia symptoms . FurtherIt is also possible that the P123H βS Tg mouse could be an ideal rodent model for examining potential disease-modifying therapies, especially more radical concepts, for DLB. As such, several potential therapy strategies are worth discussing. First, interfering with protein aggregation inhibition has been a widespread therapeutic strategy against neurodegenerative diseases, and it is noteworthy that a recent phase III Aβ immunotherapy against sporadic AD resulted in a clinically significant outcome . SimilarSecond, given that αS evolvability is manifested as neurodegeneration through the antagonistic pleiotropy mechanism in aging, suppression of antagonistic pleiotropy might be therapeutically effective : Tx2. InCollectively, the P123H βS Tg mouse model could be useful for the development of both symptomatic and disease-modifying therapies. Given recent progress of an in vitro system using induced pluripotent stem cells (IPSCs) for the therapy of neurodegenerative diseases, including DLB , the effTo summarize, the P123H βS Tg mice reproduced the many neurodegenerative features of Lewy body disease, including axonal pathology and both motor and non-motor dysfunction associated with protein aggregation. Provided with the prominent memory impairment in this mouse model, it is of great interest to determine whether the association of P123H βS with APs, including αS and Aβ, might be causally associated. According to our recent view of amyloidogenic evolvability, cross-seeding of APs, including αS and Aβ, and its negative regulation by βS, may be physiologically important for evolvability in reproduction, which could be manifested as DLB through the antagonistic pleiotropy mechanism in aging. Such concepts may be further evaluated by cross experiments of the P123H βS Tg mice with AD model mice. Furthermore, symptomatic and various disease-modifying treatments of DLB could also be assessed using these model mice, in which non-motor symptoms, such as dementia and depression, are prominent features. Thus, the P123H βS Tg mice may be a valid model for investigating the mechanism and therapy of DLB.


Walnut pellicle color is a key quality attribute that drives consumer preference and walnut sales. For the first time a high-throughput, computer vision-based phenotyping platform using a custom algorithm to quantitatively score each walnut pellicle in L* a* b* color space was deployed at large-scale. This was compared to traditional qualitative scoring by eye and was used to dissect the genetics of pellicle pigmentation. Progeny from both a bi-parental population of 168 trees (‘Chandler’ × ‘Idaho’) and a genome-wide association (GWAS) with 528 trees of the UC Davis Walnut Improvement Program were analyzed. Color phenotypes were found to have overlapping regions in the ‘Chandler’ genetic map on Chr01 suggesting complex genetic control. In the GWAS population, multiple, small effect QTL across Chr01, Chr07, Chr08, Chr09, Chr10, Chr12 and Chr13 were discovered. Marker trait associations were co-localized with QTL mapping on Chr01, Chr10, Chr14, and Chr16. Putative candidate genes controlling walnut pellicle pigmentation were postulated. Juglans regia L.) is an economically important edible seed that is diploid 2x = 32 with 16 chromosomes and a genome size of approximately 600 Mbp. In the U.S., the English walnut industry generates 1.5 billion dollars annually with over 99% of production occurring in California  and is formed from maternally derived tissue arising from the inner and outer ovular integuments (Juglans regia L.) pellicle pigmentation can vary significantly between cultivars and developmental stages, and is influenced by environmental stressors. Flavonoids or phenolic compounds synthesized through the phenylpropanoid pathway are localized in seed coats to defend the embryo against biotic stressors , and subjectively by eye using a color chart published by the California Dried Fruit Association (DFA) . Color pHuman perception of color is largely influenced by the number and types of photoreceptors an individual possesses. Differences in the photoreceptors between individuals contributes to variability in the perception of a given color. This forms the basis of subjectivity of human quality assessments within a color perception dataset. As such, color(s) quantified by human perception can be an unreliable source for obtaining quality data , howeverUsing traditional scoring, walnut pellicle color is labor intensive to measure, strongly influenced by environmental variation, and is a polygenic trait  was used for QTL mapping with a range in tree age of 8 – 12. Trees in this study were planted across eight blocks; five of them were across the same field location and all trees were spaced six feet apart and irrigated with micro-sprinklers. Blocks consisted of trees planted in row plots with multiple family crosses made in one year. A full-sib family had between 10 and 50 individuals with 31 unique parents. Most male parents were only used once, with a few male parents used 2-4 times, one male was used for 13 different crosses, and two males were used reciprocally as females.At least 30 nuts per tree were hand harvested as each tree reached fruit maturity (hull-split) during August and September of 2015, 2016, and 2017. These were hung in polypropylene mesh bags, air-dried for two weeks, and then stored at -4° until measurements were taken. Walnut kernels were placed on commercial scoring trays obtained from the Dried Fruit Association of California which contain 100 wells each. Each tray held ten kernels from each of 10 distinct trees, and 10 kernels were evaluated. When kernels were being evaluated for color, they were kept at 0° for short-term storage, and kernels were kept at - 4° for long term storage.Qualitative color scoring by eye was done by examining each kernel for its separation into color category 1 - 4, according to the Dried Fruit Association (DFA) scoring sheet , Safe F, in accoa; LITE = light, 2b; LAMB = light amber, 3c; AMBR = Amber, 4d , a* (red/green color), and b* (yellow/blue color). The data produced contains each color scores for every pellicle measured. Details on system and method was modified from  which mounts over an enclosed dome encircled with four (D65) light emitted diodes (LED) tubes of 18W  which provided consistent illumination. Custom algorithms were developed for calibration and image acquisition of the computer vision system . Briefly, The calibration macro written in FIJI 1.52 software lk is the interaction between family and year, aijk is the effect of age on color measurement for the i-th genotype, j number of measurements in the k-th year, and εijkl is the random error term with mean 0 and variance σ2ε.Multi-year phenotypic data collected for DFA scores and the CVS scores were analyzed with ANOVA using R packages ‘car’, ‘agricolae’, ‘lme4’ with equation (1) for the model,i is the effect on color measurement of the i-th genotype, βk is the effect on color measurement of the k-th year, (αβ)ik is the interaction between genotype and year, and εijk is the random error term with mean 0 and variance σ2ε. For the larger dataset of the 31 full-sib families, breeding values (BV) per individuals were estimated using ASReml-R v4 (τ is a (p × 1) vector of fixed treatment effects (block and year), X is an (n × p) design matrix, u is the (q × 1) vector of random effects (pedigree), Z is an (n × q) design matrix, e is the (n × 1) vector of residual error. The pedigree utilized in the model was reconstructed to identify corrected kinship relationships from the original UC Davis Walnut Improvement Program pedigree using the Axiom J. regia 700K SNP array  for linear model,scanone and ‘Haley Knott’ regression  and 100 imputations. A multiple QTL model was then fitted with the function fitQTL where both trait QTL identified in scanone were added in the model. The function refineQTL was applied and adjusted if the new positions in the ANOVA the model had a greater fit . The Bayesian credible interval for 5% error rate was implemented as the confidence QTL interval.Quantitative-trait loci mapping was performed using the genetic maps (File S5) of the bi-parental population ‘Chandler’ × ‘Idaho’ described in (gression . Signifigression . When muJ. regia 700K SNP array as described in (The whole population was genotyped using the Axiom ).Principal components of SNP markers were obtained by running the mixed linear model (MLM) algorithm in GAPIT. The first component explained 9.39% variation, the second PC explained 6.97% variation, the third PC explained 5.31%, the fourth PC explained 3.41%, and the fifth PC explained 2.63%. The number of principal components (PCs) to include as covariates in the models was defined based on the screeplot . This model was set to perform 10 iterations, with four to six PC’s added as covariates, depending upon the goodness of fit in the QQ-plots for each color phenotype, a minor allele frequency threshold of 5%, and the default parameters for bin size. A 5% Bonferroni threshold (2.29 × 10−7) was used to assess significance, and Q-Q plots and Manhattan plots were inspected for evidence of inflation. A multiple corrections test (−6).Genome-wide associations were conducted using the Fixed and Random Model Circulating Probability Unification (FarmCPU) algorithm . The MLM2 between 100-120 kb. Within these genic regions the predicted mapped genes and functional annotation as described in  respectively. Output tables from ANOVA in the GWAS population provided in File S4. ‘Chandler’ and ‘Idaho’ genetic maps with phenotypic data are available in supplemental (File S5). Estimated breeding value phenotypes and principal components used in GWAS analysis are available in supplemental . Supplemental data file S8 contains raw color score data for both the GWAS population and ‘Chandler’ x ‘Idaho’ population. Supplemental material available at figshare: P < 0.0) factors were family, year of harvest, family × year interaction, individual, and age; however age was not a significant factor in the linear models for DFA and L* phenotypes (File S8). For the calculation of adjusted means with ‘Chandler’ × ‘Idaho’ dataset, all terms and their interactions were significant (P < 0.0001) (File S4).In general, ‘Chandler’ produces kernels with an extra-light pellicle color, while Idaho’s kernels are light amber ; these differences are consistently observed in both the DFA and CVS analyses. For the ANOVA linear models significant . ‘Scharsch Franquette’, a selection made in California which is genetically identical to ‘Franquette’, a French variety, is a founder of the UC Davis Walnut Improvement Program, displayed the highest breeding value (47.35) for the lightness L* score. Families that possessed multiple individuals with top L* score breeding values included 10-024 (03-001-977 × ‘Ivanhoe’), 11-030 (04-003-107 × ‘Ivanhoe’) and 09-25 (95-026-16 × 94-019-85) along with founder ‘Waterloo’, and ‘Chandler’ (Table S1).Pellicle color measurements acquired using the new image-based phenotyping method yielded values of the narrow-sense heritability similar to the DFA scoring system  .Principal component analysis revealed four to six principal components which explained SNP variation for the 31 full-sib families in the Walnut Improvement Program, excluding ‘Chandler’ × ‘Idaho’ . See 2 = 0.37) and with SNP associated with the a* score on Chr16 (r2= 0.63). Pairwise LD measures between these SNPs associated with DFA and L* scores and SNPs associated with DFA and a* scores are visualized to show close proximity to each other 16.29 kb and 213 kb respectively  respectively (Table S2).Similarly, (CAD), which is associated with the b* score. Lastly, the third gene function which lies on Chr01 encodes tryptophan synthase (TRP), which is also associated with the b* score. One gene function (transcription factor MYB1) associated with the DFA score was identified on Chr07. For the a* score, on Chr10 flavonol synthase/flavanone 3-hydroxylase (FLS) was identified to be associated with a SNP, as well as on Chr11 chorismate mutase (CM). Two putative gene functions were identified on Chr12; the first of these genes was associated with both an L* and b* score, and encoded 4-coumarate—CoA ligase while the second gene encoded flavonoid 3′-monoxygenase, was associated with a DNA score. Four gene functions were identified to be associated with SNPs on Chr13; the first gene function is anthranilate phosphoribosyltransferase (PAT) which was associated with a b* score, the second gene function was also associated with a b* score and encoded caffeic acid 3-O-methyltransferase (COMT), and the final two gene functions on Chr13 both encoded FLS genes, were both associated with L* scores  putative candidate genes were identified within 100 kb windows surrounding the significant SNPs (Table S3). These putative candidate genes are predicted to have functions related to abiotic stress, pathogen resistance, hormone signaling and the biosynthesis of pigmented metabolites , indicating that these genes may contribute to a slight reddish/brown pigmentation. CM is involved in the biosynthesis of phenylalanine and tyrosine as well as their derivatives, the phenylpropanoids, lignins, flavonoids and anthocyanins, most of which are pigmented . FLS/F3H is a bifunctional enzyme , suggesting that these genes promote yellow color. PAT and TS both act in the biosynthesis of tryptophan, a light yellow AAA and its yellow/orange derivative tryptamine , suggesting that these genes are associated with a pigmentation of the pellicle; bifunctional FLS/F3H- FLS desaturates dihydroflavonols to yield yellow/brown flavonols (F3′H was associated with the DFA score (2.2), indicating a possible diversity of colored pigments. F3′H hydroxylates flavanones, dihydroflavonols, flavones and flavonols to increase the diversity of each of the aforementioned categories of yellow flavonoids  markers from the most significant SNPs, assisting in efficient selection at the seedling stage rather than the selection process over a 5-10 years period requiring trees to reach sexual maturity. Early selection of progeny will dramatically increase the efficiency in developing new cultivars. Due to the fact that walnut growers are compensated based on human grader’s perception of color, an improvement to the CVS algorithm would include human input factors such as discrimination for veining, speckling or size can be considered.For further investigation, the creation and evaluation of genomic prediction models will enable the integration of all the small QTL effects to predict progeny with lighter pellicles, as the industry demands. To solidify results pertaining to the putative candidate genes for flavonoid production, an experiment setup to clone each gene and express them in bacteria would verify gene function and identify the origin of the chemical reactions occurring in response to developmental stages, environmental stress or autophagy.