NOTCH2NLC GGC repeat expansion to investigate the pathology of retinopathy. All four NIID patients were diagnosed by skin biopsy and NOTCH2NLC GGC repeat analysis. Ocular findings in patients with NIID were studied using fundus photographs, optical coherence tomographic images (OCT), and full-field electroretinograms (ERGs). The histopathology of the retina was studied on autopsy samples from two cases with immunohistochemistry. All patients had an expansion of the GGC repeat (87–134 repeats) in the NOTCH2NLC. Two patients were legally blind and had been diagnosed with retinitis pigmentosa prior to the diagnosis of NIID and assessed with whole exome sequencing to rule out comorbidity with other retinal diseases. Fundus photographs around the posterior pole showed chorioretinal atrophy in the peripapillary regions. OCT showed thinning of the retina. ERGs showed various abnormalities in cases. The histopathology of autopsy samples showed diffusely scattered intranuclear inclusions throughout the retina from the retinal pigment epithelium to the ganglion cell layer, and optic nerve glial cells. And severe gliosis was observed in retina and optic nerve. The NOTCH2NLC GGC repeat expansion causes numerous intranuclear inclusions in the retina and optic nerve cells and gliosis. Visual dysfunction could be the first sign of NIID. We should consider NIID as one of the causes of retinal dystrophy and investigate the GGC repeat expansion in NOTCH2NLC.The retinal pathology of genetically confirmed neuronal intranuclear inclusion disease (NIID) is yet unknown. We report the ocular findings in four NIID patients with The online version contains supplementary material available at 10.1186/s40478-023-01564-3. Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells , 34, 36.NOTCH2NLC gene were reported as the cause of both the sporadic and familial NIID [Skin biopsy has been used as a safe method to diagnose NIID , 33. In ial NIID , 31, 32.Retinal damage in NIID patients has been reported , 24. SevNOTCH2NLC gene and further analysed the retinal histology of two cases by autopsy.In this study, we examined the retinal findings in four NIID patients diagnosed by skin biopsy and genetic analysis of the NOTCH2NLC GGC repeats, both with positive results. In two of these cases, consent was obtained for eye examination at autopsy, and in the other two cases, RP was diagnosed prior to the diagnosis of NIID. They were recruited from National Hospital Organization Nagoya Medical Centre, Nagoya University Hospital, and Fukushimura Hospital, from 2013 to 2021with written informed consent of all patients. The protocol of this study was approved by the institutional review board of each institute. Clinical manifestations of Case 1 were partially reported [We include four sporadic adult-onset NIID cases. All patients were assessed by skin biopsy and genetic testing of the All patients underwent ophthalmic screenings. The medical histories and the results of the clinical examinations including neurological findings, brain MRI, the best-corrected visual acuity (BCVA), slit-lamp examinations, ophthalmoscopy, ERGs, spectral domain optical coherence tomographic (SD-OCT) images, and fundus photographs were analysed. Full-field ERGs were recorded from patients who conformed to the guidelines of the International Society for Clinical Electrophysiology of Vision Standards . All patSkin biopsy was performed and assessed in the same manner as in a previous report . Cases 1For the assessment of intranuclear inclusion frequency, we selected 12 random microscopic fields of the retina. For the assessment of intranuclear inclusion frequency in the glial cells in the optic nerve and brain frontal lobe, 10 random microscopic fields were used and analysed. The numbers of the nucleus of the retinal ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), and optic nerve head, neurons and astrocytes in frontal lobe, and the number of cells with p62-positive intranuclear inclusion were counted. The ratio of the number of cells with p62-positive intranuclear inclusion per the number of cells was calculated for each image.NOTCH2NLC was assessed by repeat primed PCR, and the number of GGC repeat expansions was confirmed with fluorescence amplicon length analysis as described in detail in a previous report [Genomic DNA of peripheral blood leukocytes was extracted from NIID patients, and GGC repeat expansion in s report . In Cases report ) to scres report . About As report .NOTCH2NLC gene  and Frontal Assessment Battery (FAB) gradually deteriorated . He had repeated vomiting attacks and required multiple hospitalizations. At the age of 79, an ophthalmology examination showed that the decimal BCVAs were relatively well preserved at above 0.6 in 2 eyes  and it was difficult to determine which photoreceptor, rod or cone, was more affected  in thesMacroscopic findings of the central nervous system showed frontal dominant cerebral atrophy, and marked atrophy of white matter and ventricular dilatation were observed in both Cases 1 and 2.In the retinal section, p-62 positive intranuclear inclusions were observed in the nuclei of the retinal ganglion cell layer Fig. E: Case 1We also studied about the gliosis of retina and optic nerve Fig. . In bothHistopathological findings of the central nervous system showed that the volume and structure of hippocampus CA1 remained unchanged Fig. A, and weIntranuclear inclusions were also observed in the peripheral nervous system such as in the dorsal root ganglia Fig. H, I, symNOTCH2NLC GGC repeat expansion. Autopsy findings of Cases 1 and 2 showed widely distributed p-62 and ubiquitin-positive intranuclear inclusions. These findings were identical to the past NIID autopsy reports [All NIID cases in this study were diagnosed by both skin biopsy findings and  reports , 33, 36,NOTCH2NLC GGC expansion surely caused histopathological change and damage in the retina as well as in other nervous systems.In retina, intranuclear inclusions were observed in the entire retinal layers, from the retinal pigment epithelium to the retinal ganglion cell layer in both two autopsied cases. In addition, an observation with electron microscopy of those intranuclear inclusions in the retina revealed the same findings as the intranuclear inclusions of NIID in previous reports , 22, 34.NOTCH2NLC were not investigated. The intranuclear inclusions were found only in nervous system organs. About retina, intranuclear inclusions were found only in ganglion cells, but not in bipolar cells or retinal pigment cells in retina. Loss of ganglion cells, loss of retinal nerve fibres, and reactive astrocytosis were also reported.Only one young NIID case had been reported about the retinal histopathology that case presented dysarthria and nystagmus at the onset, age of 11 years, and examined at autopsy at the age of 21 years . About tNOTCH2NLC, such as FMR1, LRP12, GIPC1 and so on. Differences in pathological findings between previous young case and our cases may be due to the differences in genetic backgrounds between NOTCH2NLC and other genes, and such histopathological difference may be useful for differential diagnosis of these diseases in future. Further investigation in a larger number of cases is required.About our cases in this study, intranuclear inclusions were found in nervous systems and also other organs. And in retina, intranuclear inclusions were more widely observed than previous reported case, that is, not only in ganglion cells, but in inner and outer cell layers including bipolar cells and in retinal pigment epithelium cells. Differences were observed in cell types in which intranuclear inclusions were observed between our case and previous case. Some neurodegenerative diseases such as fragile X-associated tremor/ataxia syndrome (FXTAS) and oculopharyngodistal myopathy (OPDM), were reported to present similar histopathological findings of NIID , 7, 28 sNOTCH2NLC GGC repeat expansion [Marked gliosis was observed in retina and optic nerve similar to previous report. These findings suggests that gliosis was induced as a result of tissue damage to the retina and optic nerve, and these histological changes may contribute to the ocular manifestations of NIID with xpansion .The thinning of the outer nuclear layer in OCT in Case 1 seemed to be related to the accumulation of intranuclear inclusions in the outer nuclear layer cells, resulting in photoreceptor cell death, and may have affected the b-wave attenuation in the D.A 0.01. The b-wave in D.A 0.01 was known to be originated from the electrical activity of retinal outer nuclear layer bipolar cells, and the dysfunction of outer nuclear layer bipolar cells may have contributed to the reduced amplitude of b-wave in D.A 0.01 with or without impairment of the rod cells. However, the foveal microstructure of OCT was well-preserved in Case 1; therefore, such patients may not complain of any symptoms even though the OCT showed thinning retina and/or EZ disruption.In addition, the presence of intranuclear inclusions in the inner nuclear layer suggests dysfunction of the cells in the inner nuclear layer, which would also affect the amplitude of ERGs. Moreover, the peripapillary chorioretinal atrophy might be caused by retinal pigment epithelium cell dysfunction associated with the intranuclear inclusions. This pathology may bring reduced thickness of the retina in OCT and abnormal findings in the ERGs.The frequency of intranuclear inclusions in retinal cells in Case 1 were about 10–25%, which is similar to a previous NIID report on the frequency in the cerebral cortex . HoweverOn retinal degeneration, we had thought over the co-occurrence of NIID and other distinct retinal dystrophies. However, genetic analysis by WES did not identify any causative gene variants of other retinal diseases in Cases 3 and 4.NOTCH2NLC could cause severe retinal degeneration with almost non-recordable ERG and could be clinically diagnosed as retinitis pigmentosa. One study reported that there was no significant correlation between the lengths of the GGC repeats and the age of onset or severity of the disease [NOTCH2NLC GGC repeats, further studies in more NIID cases are necessary.This result indicated that the expansion of the GGC repeat of the  disease . HoweverIntranuclear inclusions diffusely existed in the entire retina without preference of cell types. This condition may lead to a variety of retinal clinical symptoms and findings including retinal pigment epithelium atrophy, photoreceptor death, bipolar cell dysfunction, or optic atrophy. Additionally, the way of retinal degeneration may differ from those of conventional dystrophies.Focusing on Cases 3 and 4 with severe retinal degeneration, unlike typical RP, there was a greater propensity for macular involvement. In some previous studies, NIID-related retinopathy showed a peculiar pattern of chorioretinal atrophy which started from the peripapillary region and extended to the macula or the temporal arcade , 11, 24.SCA7, SCA1, SCA3 and Huntington's disease, polyglutamine diseases caused by expanded CAG repeats, have been reported to cause clinical and experimental retinal degeneration , 26, 38.SCA7 and NIID are partially similar in that the distribution of intranuclear inclusions and the site of neuronal loss do not necessarily coincide. This phenomenon may indicate that the expanded repeats sequence brings toxic function, but the intranuclear inclusions visible by light microscopy are not themselves toxic. The molecular mechanisms leading to the discrepancy between intranuclear inclusion distribution and neuronal loss may be similar between SCA7 and NIID. Focusing on this point, it is necessary to elucidate the molecular mechanism of cytotoxicity in these diseases.Several studies have reported ophthalmologic findings of patients with NIID, ranging from reduction of the ERG signals in the absence of subjective symptoms to severe retinal degeneration resulting in legal blindness , 39, 40.NIID has been reported to present a very wide distribution of intranuclear inclusions in cells of the central nervous system, peripheral nervous system, and non-neurological tissue. Yet, the distribution of intranuclear inclusions and cell loss did not correlate well. Moreover, neurological manifestations of NIID did not correlate with the distribution of intranuclear inclusions, and oftentimes, intranuclear inclusions were observed in more extensive sites compared to what was expected from clinical manifestations , 29, 36.Because the retina and optic nerve are part of the nervous system, there may be an imbalance between the distribution of intranuclear inclusions in ophthalmologic tissues and ocular symptoms. Additionally, electrophysiological examinations showed delayed conduction velocity and reduced amplitudes in NIID cases , 33. SucCase 1 presented miosis, and autopsy findings showed almost normal findings of the Edinger-Westphal nucleus that supplies parasympathetic fibres to the eye and constricts the pupil, but many intranuclear inclusions and neuronal loss were observed in the sympathetic ganglia. This pathological imbalance of the damaged sympathetic ganglion and almost intact Edinger-Westphal nucleus may bring parasympathetic nerve dominancy about the pupil diameter, and may be resulting in miosis.This study has some limitations. One is the incomplete examinations in the asymptomatic patients. We have experienced patients with NIID who were ophthalmologically asymptomatic or who had minimal fundus findings. Although detailed analysis of the retina is required by visual field tests and wide-angle image analysis, most of such patients did not want further ocular examinations. A second limitation is the small number of patients which was probably because that NIID is not yet widely recognized as a disease in the field of ophthalmology. Our cases included sporadic elderly patients, but it is also necessary to confirm whether there are changes in the retina in family cases and/or in younger cases. Third, this study was a cross-sectional study, and it is possible that even patients with no subjective visual symptoms may develop impairment of their visual function from a progression of NIID. Long-term studies of the retinal morphology and function are essential even for NIID patients without visual symptoms.NOTCH2NLC GGC expansion. These results suggested a unique pathology of retinal degeneration in NIID and heterogeneity of ocular manifestations in NIID related to the variety of affected cell types. Autopsy reports of retinal histology in NIID patients are rare. The pathogenicity of NIID is probably different from conventional hereditary retinal diseases. Our findings indicate that visual dysfunction could be the first sign of NIID. We should consider NIID as one of the causes of retinal dystrophy, especially showing an atypical progression of retinal degeneration, and investigate the GGC repeat expansion in NOTCH2NLC.In conclusion, we confirmed the diffusely scattered distribution of intranuclear inclusions in the retina, gliosis in retina ant optic nerve, and the diversity of the retinal abnormalities in patients with NIID with Additional file 1: The summary of SNV in RetNet genes.


Thoracic endovascular repair (TEVAR) is currently the recommended and most widely used treatment for type B aortic dissection. A major challenge is revascularization of the left subclavian artery in order to extend the landing zone to zone 2 (Ishimaru classification). Various strategies have been used for revascularization, including branched stent graft, fenestrated stent graft, the chimney technique, the parallel technique, and bypass surgery. Single-branched stent graft is one of the most promising strategies, and several products have recently been reported as potential candidates for use with this approach. The Castor single-branched stent graft is the only off-the-shelf product available; this product has been developed through collaboration between Chinese corporations and clinicians. In this Perspective article, clinical experience and data obtained from TEVAR with the Castor single-branched stent graft are summarized by experienced Chinese experts. Without treatment, the mortality rate of acute aortic dissection will exceed 22.7% within 6 h, 50% within 24 h, and 68% within the first week . The manIn patients with TBAD, the primary entry tear typically originates a few centimeters distal to the ostium of the left subclavian artery (LSA) owing to the large fluctuation in dP/dt pressure at the curvature , 5. Msea2.Branched stent graft is one of the most commonly used strategies to preserve the LSA (the other being fenestrated stent graft). Several products have been reported as offering potential solutions for complete sealing in zone 2 while keeping the LSA patent; these include the Valiant Mona left subclavian artery (LSA) stent graft  , the Gor3.3.1.The Castor single-branched stent graft has a unibody design, which means that there is no gutter or space between the branch section and the main body. The unibody design of this stent graft could greatly reduce the risk of endoleak compared with the parallel stent graft (type Ia) and fenestrated stent graft (type II), and its multifilament PTE graft could also reduce the risk of type IV endoleak.The ostium of the LSA can originate from various clock positions of the aortic arch. Yoon et al. found that the median clock position of the ostium of the LSA was 12:00 , and Mougin et al. found that the median clock position of the ostium of the LSA relative to the ostium of the LCCA was −10 min (−30 to 0 min), suggesting low variability in the relative position of the LSA , 17. The3.2.The Castor single-branched stent graft is specifically designed to align with the morphological characteristics of TBAD. According to the Instructions for Use (IFU), its indications include (1) thoracic aortic dissection retrograde to the LSA and (2) thoracic aortic dissection requiring proximal fixation in zone 2 and zone 3.It has been well recognized that TEVAR should be considered for complicated hyperacute, acute, and subacute TBAD patients and for uncomplicated hyperacute, acute, and subacute TBAD patients with high-risk characteristics , 19. WheIn addition, there are some reports on the use of the Castor single-branched stent graft in cases of zone 1 TEVAR: the LCCA is revascularized with the branch section, and the LSA is revascularized by creating an extra fenestration in the main body. This strategy is off-label and lacks verification, and should therefore be considered with great caution.3.3.Computed tomography angiography (CTA) images and a workstation with centerline luminal reconstruction are necessary for precise CTA evaluation. The diameters of the LSA, true lumen, false lumen, and estimated true lumen at the estimated proximal and distal landing zone are measured to determine the proximal diameter of the stent graft, its taper ratio, and the length of the main body. The distance between the ostium of the LCCA and the LSA at the side of greater curvature is measured to determine the distance between the branch section and the proximal edge of the main body. The diameter of the LSA is measured to determine the diameter of the branch section. The distance between the ostium of the LSA and the ostium of the left vertebral artery (LVA) is measured to determine the length of the branch section, where coverage of the ostium of the LSA should be avoided. When the taper ratio of the main body cannot meet the requirements of the lesion, a distal-restrictive stent graft can be considered to avoid aortic rupture.3.4.in vitro, and it was found that a force of 24N was required in order for the Castor single-branched stent graft to migrate, which was twice that required in the case of the straight stent graft. Since the branch section could serve as an anchor to ensure fixation, the radial force generated from the oversized stent graft compressing the aortic wall could be reduced. Thus, an oversize ratio of 0%–5% for the proximal landing zone is acceptable for the prevention of migration, while ensuring tight adherence to the aortic wall to avoid type Ia endoleak.The suggested oversize ratio for aortic aneurysm is 20%; however, for aortic dissection, and especially for TEVAR with a single-branched stent graft, the oversize ratio should be reconsidered. An oversize ratio of 0%–5% for the proximal landing zone has been applied for endovascular repair of TBAD at many centers in China, which could greatly reduce the radial force of the stent graft on the fragile aortic wall. For a straight stent graft, the radial force is the main source of fixation. In the Castor single-branched stent graft, the unibody design allows the branch section to function as an anchor, thus providing extra strength for fixation. The fixation abilities of a straight stent graft and the Castor single-branched stent graft were compared 4.4.1.The femoral artery (left or right) is the recommended conduit for delivery of the stent graft, and the left brachial artery (LBA) is the recommended conduit for access to the left subclavian artery. Once the LBA is cannulated, a catheter is advanced through the guide wire from the LBA to the exposed femoral artery to build the conduit for the branch section. After withdrawal of the guide wire within the conduit, the traction of the branch section is inserted into the conduit (catheter) from the femoral artery and acquired from the LBA.4.2.An extra-stiff guide wire  pigtail catheter is advanced into the ascending aorta from the femoral artery, and a pigtail catheter is advanced through the extra-stiff guide wire into the aortic arch. After withdrawal of the extra-stiff guide wire, the C-arm machine is set at the desired angle according to the preoperative measurement, such that all branches of the aortic arch can be viewed clearly, mostly between the left anterior oblique 30° and 60°. After these preparatory procedures, the initial angiography is performed to evaluate the morphological characteristics of the aorta and lesions.4.3.When angiography is complete, the extra-stiff guide wire should be inserted into the ascending aorta again and the pigtail catheter removed. The single-branched stent graft is then advanced over the extra-stiff guide wire into the thoracic descending aorta with the traction wire of the branch section simultaneously drawn at the point of the LBA. The outer sheath is removed and retained in the thoracic descending aorta, and the inner sheath (which is softer than the outer sheath), together with the stent graft, should be advanced into the aortic arch. After arrival at the target position, the inner sheath is removed and the branch section is dragged into the LSA by pulling the traction wire of the branch section. The main body of the single-branched stent graft is deployed by withdrawing the trigger wire, and the branched section is subsequently deployed by withdrawing the traction wire. After deployment of the single-branched stent graft, further angiography should be performed to verify complete exclusion of the lesion without endoleak, and to confirm that the LSA is patent .4.4.Use of the direction inversion technique during deployment could lower the risk of misalignment between the branch section and the LSA. The clock position of the ostium of the LSA is within the range of 11:00–1:00 o’clock. When entering the femoral artery, the direction of the branch section should be pointed toward the floor, where the branch section will turn toward the direction of the ostium of the LSA at the aortic arch.During advancement of the stent graft, rotation of the delivery system should be avoided. Even when no rotation occurs, there is a possibility that the traction wire of the branch section could become wrapped around the trigger wire of the main body. Therefore, before withdrawing the outer sheath, the operator should reconfirm that the traction wire and trigger wire are separated, or the operator should unwrap the guidewire by gently rotating the delivery system.Although the branch section can be rotated by up to 150° in multiple directions while remaining patent, the operator should avoid rotation and misalignment of the branch section and the LSA through precise preoperative measurement and careful operation. When stenosis or twisting of the branch section occurs, this can be noted using angiography. In this situation, post-dilation should be considered as a solution.5.Antiplatelet therapy should be considered in cases where the diameter of the branch section is <8 mm in order to prevent stenosis and occlusion of the branch section. Life-long surveillance is required after TEVAR with a single-branched stent graft. Contrast-enhanced CT is recommended at 1 month, 6 months, and 12 months and then annually after TEVAR for TBAD. For patients with contraindications to iodinated contrast agents, duplex ultrasound and non-contrast CT scan are recommended at 1 month, 6 months, and 12 months and then annually after TEVAR for TBAD.6.An off-the-shelf single-branched stent graft simplifies the procedure for TEVAR with the requirement for LSA revascularization. With a steadier and shorter learning curve and a high success rate, the Castor single-branched stent graft is expected to become an important off-the-shelf option for endovascular repair of TBAD with insufficient healthy landing zone.


Zeaxanthin is a natural xanthophyll carotenoid and the main macular pigment that protects the macula from light-initiated oxidative damage, but it has poor stability and low bioavailability. Absorption of this active ingredient into starch granules as a carrier can be used to improve both zeaxanthin stability and controlled release. Optimization using three variables judged important for optimizing the system  was conducted for incorporation of zeaxanthin into corn starch granules, aiming for high zeaxanthin content (2.47 mg/g) and high encapsulation efficiency (74%). Polarized-light microscopy, X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared spectroscopy showed that the process partially gelatinized corn starch; additionally, it showed the presence of corn starch/zeaxanthin composites, with the zeaxanthin successfully trapped in corn starch granules. The half-life time of zeaxanthin in corn starch/zeaxanthin composites increased to 43 days as compared with that of zeaxanthin alone (13 days). The composites show a rapid increase in zeaxanthin release with in vitro intestinal digestion, which is favorable for possible use in living systems. These findings could have application in designing effective starch-based carriers of this bioactive ingredient with enhanced storage stability and improved intestines-targeted controlled-release delivery. Zeaxanthin  is a xanthophyll carotenoid found in egg yolks and in very small amounts in dark-green leafy vegetables such as spinach; along with lutein, it is the principal carotenoid in the lens and macular region of the retina . EpidemiDue to its biodegradability and biocompatibility, starch is an excellent material for encapsulation of bioactive compounds in the food and biomedical fields . ComparaVarious starch systems have been developed for encapsulation, including native starch granules, microporous starch, starch nanoparticles, substituted starch, cross-linked starch, hydrolyzed starch, amylose inclusion complexes, and other forms of modified starch . Among tOpuntia monacantha mucilage against the effects of light, heat, and oxygen, resulting in improved zeaxanthin retention during 28 days of storage [Zeaxanthin has been encapsulated into  storage . The enh storage . However storage .Although the effect of reaction temperature on the encapsulation of lutein into starch granules has been reported, there is as yet no detailed investigation on how other necessary reaction parameters (such as starch concentration and reaction time) affect the formation and structural characterization of corn starch/zeaxanthin composites. Such an investigation could lead to improved functional properties (storage stability and release behaviors). Therefore, in this study, the effects of reaction temperature (50–70 °C), starch concentration (2–10%), and reaction time (0.5–4 h) on the zeaxanthin content and encapsulation efficiency of corn starch/zeaxanthin composites were explored. The corn starch/zeaxanthin composites so obtained were characterized by light microscopy (LM), polarizing microscopy (PM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC), and their functional properties (storage stability and release behaviors) were examined. This study can be used to indicate how conditions can be optimized in synthesizing composites using native starch granules to deliver sensitive bioactive ingredients as functional food supplements.Native corn starch granules (amylose content ~28%) were kindly provided by Starpro Starch Co., Ltd. . Zeaxanthin (purity ≥ 85%) was purchased from Macklin Biochemical Co., Ltd. . Pancreatin from porcine pancreas  and amyloglucosidase  were purchased from Sigma-Aldrich Pty. Ltd. . All other chemicals were of reagent grade and used as received.w/v, dry basis) were prepared in deionized water, then heated in a water bath at various temperatures  under constant gentle stirring for 1 h. Ethanol (100 mL) containing 20 mg zeaxanthin was added dropwise to the resulting starch dispersions over a 30 min period under constant stirring. After incubation at various temperatures and times , the mixture was slowly cooled to room temperature. The resulting suspension was centrifuged at 3000× g for 20 min, and the precipitate was washed three times with ethanol solution  to remove unencapsulated zeaxanthin. The corn starch/zeaxanthin composites so obtained were oven-dried at 40 °C for 24 h and passed through a 200-μm nylon sieve for further use.Corn starch/zeaxanthin composites were prepared following a method described elsewhere ,14 with g for 10 min, and the zeaxanthin in the supernatant was analyzed from its absorbance at 458 nm [Each sample of the corn starch/zeaxanthin composites  was completely dispersed in 3 mL of dimethyl sulfoxide (DMSO) by vigorous vortexing. The resulting mixture was centrifuged at 3000× t 458 nm . The zeaThe encapsulation efficiency is reported as the percent of zeaxanthin recovered from a composite in relation to the total zeaxanthin added during its formation as follows:w/v, dry basis) sample suspension in the aqueous glycerol solution (1:1 water/glycerol) was characterized using an Olympus BX53  with brightfield and polarized light [A 1% (ed light .For scanning electron microscopy (SEM), samples were mounted on aluminium stubs with double-sided sticky tape and sputter-coated with a thin film of gold using a Hitachi S-4800Ⅱ scanning electron microscope  operating at 10 kV accelerating voltage and 4 mm working distance with 500× magnification .The XRD analysis of samples was carried out using an X-ray powder diffractometer , operated at 40 kV and 40 mA. Samples were manually packed tightly onto the glass sample holder, and data collected over an angular range from 2θ = 4° to 35° with a step of 0.02° . Their rw/w) manually in an agate mortar for 1 min, and then the above mixed powders (0.2 g) were pressed into pellets of 13 mm diameter and 1 mm thickness under 10 MPa using an automatic tablet press for analysis [−1 using an Antains II FTIR spectrophotometer  [Samples were ground with KBr  .d = 2π/q, where d (nm) is the lamellar repeat distance and q (nm−1) is the scattering vector, with q = (4π sinθ)/λ, where λ (nm) is the X-ray wavelength and 2θ is the scattering angle.Samples were mixed with deionized water to achieve ~60% of total moisture content and equilibrated at ambient temperature (~25 °C) for 24 h. A small-angle X-ray scattering diffractometer , equipped with a Vantec 2000 detector  and pinhole collimation for point focus geometry, were used for sample lamellar structure analysis, as described elsewhere . The aveSamples  were mixed with distilled water to achieve 70% total moisture content and hermetically sealed in stainless steel pans for 12 h to equilibrate. The thermal properties of samples were analyzed by differential scanning calorimetry  with a scanning range from 30 °C to 150 °C at a rate of 10 °C/min .k) and half-life time (t1/2) for the retention of zeaxanthin were calculated from these plots as ln (zeaxanthin retention) = a − kt and t1/2 = ln 2/k [Residual zeaxanthin content (mg/g) in the samples was analyzed over 21 days of storage, and the zeaxanthin retention  was plotted against storage time on a semi-log plot. The data are interpreted assuming simple first-order loss. The rate coefficient . Significant differences in the data were analyzed by analysis of variance (ANOVA) with Duncan’s multiple range test at the XRD diffraction patterns of zeaxanthin, corn starch, and corn starch/zeaxanthin composites are shown in −1 and the peak at 964 cm−1  is the lamellar repeat distance  and q (nm−1) is the scattering vector. Corn starch shows a characteristic peak at about q = 0.65 nm−1  of corn starch granules, consistent with the literature [1 and c2) were observed at q = 0.35 nm−1 and 0.46 nm−1 for the corn starch/zeaxanthin composites (1 and c2)), corresponding to two semicrystalline structures with d of 17.95 nm and 13.66 nm in the composites, respectively. The first peak (q = 0.35 nm−1) in the corn starch/zeaxanthin composites should be closely related to the formation of amylose/zeaxanthin V-type complexes [q = 0.46 nm−1) is attributed to starch retrogradation after insufficient gelatinization, resulting in less disordering occurring after the encapsulation [The rrection  is preseterature . Two newomplexes , and thesulation .To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy (ΔH), are presented in To, Tp, Tc, and ΔH compared with corn starch, indicating more higher crystallinity, consistent with the SAXS data. A similar observation was reported in the starch/zein nanocomposites, which exhibited a thermogram shifted to higher temperatures due to molecular interactions between the starch chain and zein [The thermal properties of zeaxanthin, corn starch, and corn starch/zeaxanthin composites, namely the onset temperature . This compares with the retention of zeaxanthin in Opuntia monacantha mucilage-based nanoparticles and nanoemulsion of 46% and 30%, respectively, after 21 days of storage at 25 °C [at 25 °C . This shTo  show the presence of both amylose/zeaxanthin complexes and retrograded starch after insufficient gelatinization, all of which would be expected to contribute to enhanced storage stability (76.1 °C, . Corn sttability  and intetability  of zeaxaThis study investigated the formation, structures, and functional properties of composites between native corn starch granules and zeaxanthin, aiming for the encapsulation of zeaxanthin as an active ingredient in corn starch as an inert carrier. The optimal conditions achieved here were a zeaxanthin content of 2.47 mg/g and encapsulation efficiency of 74.00% in the corn starch/zeaxanthin composites, at a reaction temperature of 65 °C, 6% starch concentration, and 2 h reaction time. The resulting corn starch/zeaxanthin composites displayed a rough appearance with thin lamellar structures and enhanced V-type starch crystallinity. FTIR confirmed that zeaxanthin was successfully entrapped in the corn starch granules. XRD, SAXS, and DSC data together provided evidence that the corn starch/zeaxanthin composites consisted of amylose/zeaxanthin complexes and retrograded, but incompletely gelatinized, corn starch. Encapsulation of zeaxanthin into corn starch granules could protect zeaxanthin from storage degradation and achieve intestinal-targeted delivery. These could have applications in designing effective starch-based carriers of bioactive ingredients with enhanced storage stability for intestinal-targeted delivery, which would be beneficial for their potential use in the sensitive food supplement.


The evolutionary shift from a single-element ear, multi-element jaw to a multi-element ear, single-element jaw during the transition to crown mammals marks one of the most dramatic structural transformations in vertebrates. Research on this transformation has focused on mammalian middle-ear evolution, but a mandible comprising only the dentary is equally emblematic of this evolutionary radiation. Here, we show that the remarkably diverse jaw shapes of crown mammals are coupled with surprisingly stereotyped jaw stiffness. This strength-based morphofunctional regime has a genetic basis and allowed mammalian jaws to effectively resist deformation as they radiated into highly disparate forms with markedly distinct diets. The main functional consequences for the mandible of decoupling hearing and mastication were a trade-off between higher jaw stiffness versus decreased mechanical efficiency and speed compared with non-mammals. This fundamental and consequential shift in jaw form–function underpins the ecological and taxonomic diversification of crown mammals.This article is part of the theme issue ‘The mammalian skull: development, structure and function’. Among the most dramatic macroevolutionary transformations leading up to the Cenozoic diversification of the y joint) ,2. This y joint) –8. Studyy joint) ,10.Although past research focused heavily on the implications of this transition for the mammalian ear, this evolutionary transformation also affected jaw mechanics. Historically, we understand vertebrate feeding systems by characterizing the morphological components as lever arms and force transmission linkages . In this framework, the fulcrum of the jaw mechanical system (jaw joint) underwent three major transformations during the approximately 440 Myr history of jawed vertebrates. First, the evolution of an upper–lower jaw articulation in the earliest gnathostomes  enabled Here, we demonstrate that shape evolution of the mammalian mandible is characterized by a concomitant increase in morphological disparity but decrease in functional disparity, representing a form–function decoupling. Furthermore, the mammalian jaw transformation represents a trade-off between strength and efficiency; the radiation of mammalian mandibular shape diversity is coupled with higher jaw stiffness but lower jaw speed and mechanical advantage (MA) compared with non-mammals. We leverage an integrative array of analyses using elliptic Fourier transforms, experimental mechanical testing, finite-element modelling, gene–phenotype–biomechanics association analyses, adaptive landscapes and phylogenetic comparative methods on a jaw shape dataset of 1000+ vertebrates Momocs R package [X  and Y  axes using the plotting function in R. The resulting warped shapes  were exported as vector graphics files both for linear biomechanical trait measurements and for 3D bending experiments and finite element (FE) simulations (see below).We used 2D outline shape analysis to quantify vertebrate jaw shape ,25–30 f. To capt package . A serie (b)Two linear biomechanical traits based on beam theory and lever mechanics principles were measured for each of the 100 theoretical warp models from the morphospace: aspect ratio (AR) and MA. Mechanical lever ratios have been shown to overestimate force transmission and underestimate velocity transmission . TherefoWe estimated additional biomechanical traits using FE simulation and validated our simulation dataset using experimental mechanical tests. The 100 theoretical warp models were converted from vector graphics files into extruded (with a thickness of 10 mm) models that were then 3D printed for experimental bending test and digitally solid-meshed for FE simulations, respectively. For experimental bending tests, hypothetical warp models were printed and mounted onto a custom-fitted vice grip attachment to a Mark-10 ESM1500 electromechanical testing frame . Cantilever bending tests were performed on each plastic jaw model by fixing the jaw joint/grip cube and applying a ventrally directed force at the anterior tip of the model. Two mechanical property values were collected from each 3D printed model experiment: total deflection (up to 10 mm) and strain energy (SE). Deflection is a measure of the stiffness of a particular jaw shape and is measured as the vertical distance travelled by the testing frame during a given experiment. SE is another measurement of stiffness or resistance to deformation, defined as the work done in deforming a material under a particular load, and was estimated as the area under the force–deflection curve by calculating the integral of the curve (in N mm).For simulation-based bending tests using the FE method, the extruded jaw shape models (in STL format) were imported into Strand7 finite-element software  and set to have identical dimensions to the physical models tested using bending experiments. All jaw models were assigned a homogeneous set of material properties suitable for linear static analysis; the elastic (Young's) modulus was set to 20 GPa, and Poisson's ratio to 0.3, as in previous FE studies of jaw structures ,34. Noda (c)interp R package. Double linear (or bilinear) interpolation sequentially applies linear interpolation to two variables  in predicting a third (i.e. z). The biomechanical values obtained from linear measurement, experimental and FE analyses were set as the z values for the theoretical warp models and used to interpolate z values for per-species biomechanical trait in the taxon-specific shape dataset. The resulting mean interpolated values for each genus were used in all subsequent analyses.We estimated taxon-specific values of AR, MA, total deflection (stiffness) and SE (another measure of stiffness) using double linear interpolation, implemented in the  (d)To evaluate the assumption that the simplified lateral/2D profile jaw outlines used in this study retain significant functional morphological signal relative to a null model of randomly distributed shape and biomechanical traits, we conducted a series of non-parametric trait distribution analyses using a combination of bootstrap resampling and Wilcoxon rank sum tests. We first generated 1000 datasets of 1000 combinations of PC1 and PC2 values within the jaw shape morphospace as simulated taxa. The biomechanical traits  were then estimated for simulated taxa using the same interpolation formulae as those used to estimate actual taxa values. Lastly, Wilcoxon rank sum tests of similarity between the distributions of both shape and biomechanical trait values in the simulated shape dataset versus values interpolated for actual taxon shapes were conducted. The hypothesis tested here is that both the PC1–PC2 and biomechanical trait values in the taxonomic dataset collected were significantly different from randomly sampled distributions of those values in the same morphospace, and thus provide support for the 2D approach. (e)dispRity R package [To examine potential differences in jaw shape and biomechanics trait disparity between mammals and non-mammals predicted by a ‘key innovation’ hypothesis , we calc package . In addi (f)10-transformed prior to further analysis. The relationships between each of five functional morphological traits  and log10(body mass in grams) were examined using scatterplots. The relationship between log10(body mass) and total jaw adductor was plotted and then linearly regressed using the ‘power trend line’ function in Microsoft Excel.To assess the potential correlation between the morphological and biomechanical traits analysed and body mass, we collected both body mass and jaw adductor muscle mass data from a range of mammals and other vertebrates. Average adult body masses for 411 mammal genera were extracted from the PanTHERIA database  (electro (g)W is the combined adaptive landscape, nw is the per-performance surface relative fitness and nF is the individual performance surface [W is optimized as the likelihood of combinations of performance surface and relative fitness, under the definition that the total fitness sums to 1 and the variance of all surfaces is set to be equal. Using experimental SE, relative bite force (MA), bite speed (1/AR) and jaw displacement (AR) traits as performance variables, adaptive landscapes were optimized for all gnathostomes at five taxonomic levels: non-tetrapods versus tetrapods; between synapsids and sauropsids; and within synapsids, sauropsids and archosaurs, respectively. The optimized adaptive landscapes were then used as hypotheses for testing models of trait evolution.We contextualized the evolutionary and biomechanical optimality of vertebrate mandibles using adaptive landscape analysis ,45. Brie surface . For eac (h)paleotree R package [motmot R package to test the results from the adaptive landscape analyses: these models are (i) Brownian motion , (ii) Ornstein–Uhlenbeck , and (iii) ACDC .To examine evolutionary rates and modes of biomechanical traits , we constructed a composite phylogeny including extant and fossil taxa using existing literature . We also estimated branch lengths and their uncertainty over the phylogeny by generating 100 time-tree samples using the Paleobiology Database and the  package . Using ttraitMEDUSA method implemented in motmot. This rate shift detection method first fits a BM model to the data, then systematically fits two-rate models to each node in the phylogeny, and then identifies the best-fitting two-rate model using Akaike information criterion (AIC). We used this approach to test the hypothesis of a rate shift occurring near the non-mammal to mammal transition but without specifying an exact node, by allowing the fit to be searched over all possible nodes. Because of model misspecification and other biases associated with using individual multivariate shape trait variables such as individual PC axis scores for univariate comparative analysis [Furthermore, to assess whether there is a significant change in the rate of biomechanical evolution from non-mammal vertebrates to mammaliaforms during the transition to the mammalian mandible, we modelled biomechanical evolutionary rate heterogeneity using the analysis ,49, we canalysis . Pairwisanalysis . We usedTo select the best-performing biomechanical trait evolution model, we used weights from AICc  scores of the three different trait models to identify the preferred model for each of the three biomechanical traits analysed. To identify locations of potential evolutionary rate change in biomechanical traits on the composite phylogeny, we summarized the results of the two-rate model-fitting analyses as a percentage of the 100 tree samples in which a rate increase or decrease is found at a particular node. The same 100 tree samples were used to estimate magnitude and significance of multivariate evolutionary rate differences between mammal and non-mammal jaw shapes. (i)Homo sapiens as the query sequence in the BLAST search engine. A summary of the gene families and groups involved in jaw development is included in the electronic supplementary material.We conducted a relative evolutionary rate analysis using GenBank sequences of genes that have been shown to be involved in normal mandible development in vertebrates. Nineteen genes were identified from the literature as definitely expressed in the lower jaw. Reference sequences of these genes were obtained using dendextend R package [RERconverge R package for relative evolutionary rate analysis [We first assessed the extent to which the candidate genes to be used in relative evolutionary rate analyses display high degrees of covariation with each other. We used a phenetic approach (neighbour-joining) to build individual gene trees from FASTA sequences. All possible pairwise comparisons of the 19 jaw development candidate genes were then conducted by pruning each pair of gene trees to the same ingroup and outgroup taxa. The trees then were compared using the entanglement algorithm implemented in the  package . We thenanalysis . Three c (j)lavaan package) to estimate path coefficients, which provide measures of the amount of variance explained by individual traits contributing to genetic and biomechanical latent variables, respectively.To examine the correlation between genetic and jaw shape distances among jawed vertebrates, we used structure equation modelling  to quantify gene sequence covariation with multivariate jaw shape covariation and biomechanical traits. We treated the combined effects of the 19 identified jaw development genes as a genetic latent variable on which the jaw shape, represented by the pairwise Euclidean distance of all principal component shape scores among all taxa for which genetic information is available, depends. We created a second latent variable representing jaw biomechanical performance as measured by AR, MA and SE; this latent variable is correlated to jaw shape in our model, representing structure–function linkage. We fitted this structural equation model to the genetic, shape and biomechanical data using maximum likelihood in R (. 3 (a)a). The x-axis (PC1) represents a gradient of shallow  jaw shapes with low adductor processes on the negative axis to dorsoventrally deep jaws with high adductor (coronoid) processes on the positive axis. The y-axis (PC2) mainly represents a gradient of jaw shapes with shallow dorsoventral depth at the adductor process on the negative axis to those with deep dorsoventral depth at the adductor process on the positive axis. The lower left-hand corner of the morphospace  contains a region represented by biologically impossible shapes ; several taxa possess jaw shapes that border those impossible regions, but none falls within it. Such intersecting outlines are a common occurrence in elliptic Fourier analyses and are treated herein as in other studies as geometrically and biologically impossible configurations and removed from further analysis [x-axis compared with the y-axis. Generally, the major gnathostome clades overlap each other, and most clades sampled have representative taxa near the origin. Clades such as pterosaurs and squamates contain clusters of taxa that fall near the edge, representing jaw shapes with high velocity .The bivariate plot defined by the first two PC axes  together represents 59.1% of the total variance in the jaw shape dataset a. The x-analysis ,53. Data (b)MA and AR values obtained from the linear measurements are included in electronic supplementary material, data S6. Experimentally measured total deflection and SE values were collected for 93 out of 100 warped theoretical jaw shape models; seven of the theoretical models were biologically impossible and were not tested. Both deflection and SE values show a similar pattern across the morphospace: the jaw shapes with the lowest deflection (i.e. stiffest) and SE values (i.e. most work-efficient) are in the upper right corner of the morphospace . Displacement and SE values both increased with decreasing PC1 and PC2 values. Models bordering biologically impossible forms were flexible and did not fall within the parameters of the cantilever bending experiments, and thus did not have displacement or SE values. Given the strong correlation between deflection and SE values, we used the experimentally calculated SE values and maximum deflection values interchangeably to represent overall structural stiffness in adaptive landscape, trait evolution, relative evolutionary rate and latent variable path analyses.−0.284×Displacement, R2 = 0.4728), reflecting the rapid drop in stiffness of the jaw structure as AR shifts towards higher jaw closing velocity .FE simulations of cantilever bending across the 93 theoretical warp models that represent shapes at regular intervals across the all-mandible morphospace returned highly correlated maximum vertical deflection values between models with intramandibular joints and those without. Outputs of these computational models also correlated strongly to experimentally derived measurements . The lower left-hand edge of the morphospace revealed large discrepancies between simulated and experimental data . Such discrepancies stemmed from jaw shapes with very low AR (=high jaw closing velocity). Jaw deflection values in experimental data increased exponentially relative to change in AR towards thinner jaws (AR = 0.5987 × 10 (c)Wilcoxon ranked sum tests between bootstrapped null distributions and actual measured  or interpolated  taxon-specific trait values showed significant differences. All of the taxon-specific attributes are significantly different from a model of random distribution. There are more occurrences of lower MA and lower AR values in sampled taxa than expected from a random distribution of jaw shapes in the morphospace. There are more higher SE and deflection (both indicating lower stiffness) values in sampled taxa compared with randomized jaw shape samples . PC1 scores  show a distinct bimodal distribution on either side of the origin  compared with a random distribution model. On the other hand, PC2 scores exhibit a unimodal distribution around the origin rather than a random distribution. Pairwise Wilcoxon tests for between-clade differences in biomechanical trait values were conducted ; however, we caution against overinterpreting comparisons between clades that have very small sample sizes . Instead, we focus our discussion on testing mammal versus non-mammal differences. (d)Mammals exhibit significantly lower disparity in combined PC values compared with non-mammals but have significantly higher disparity specifically in PC1 and PC2 values. In addition, mammals have significantly lower disparity in all three biomechanical traits  from PC1 and PC2 theoretical warp models compared with non-mammals . Mammals exhibit significantly higher ARs (deeper jaws) and higher stiffness  than non-mammals, but lower MA compared with non-mammals .Table 1 (e)R2 was smaller than 0.01. Furthermore, linear regression analysis between body mass and total adductor mass suggests that there is no differential allometry for adductor mass to body mass scaling in the mammals (57 spp.), birds (48 spp.) or squamates (6 spp.) sampled. The squamate slope is lower (i.e. jaw adductor mass increases slower with body mass) than in either birds or mammals, but the small sample size from the squamate dataset precludes a proper statistical comparison .None of the scatterplots indicates a correlation between body mass and biomechanical and morphological traits; in all analyses the  (f)Although each taxonomic group returned a unique landscape, pairwise comparisons found significant differences only between synapsids  and sauropsids , and between turtles and all other sauropsids. Our analyses indicated no differences between non-tetrapods and tetrapods, among archosaurs, among non-testudine sauropsids or along the grade of synapsids leading to mammals.Two primary adaptive regimes were responsible for these differences: (i) a strength regime that is primarily adapted for reducing SE (i.e. increasing stiffness) and only small contributions from bite force, bite speed and jaw compliance (low AR); and (ii) a displacement regime primarily adapted for jaw compliance with moderate contribution from bite force, and small contributions from bite speed and SE. The strength regime provides generally high adaptive performance for a majority of morphospace, to which morphologically diverse groups are well adapted. Non-tetrapod vertebrates, stem tetrapodomorphs, turtles and synapsids are generally broadly distributed through morphospace and are best adapted to the strength regime, while lepidosaurs and archosaurs are generally more restricted in morphospace and are best adapted to the jaw compliance regime . Based on these findings, we hypothesize that an evolutionary rate shift in biomechanical traits occurred in lepidosaurs and archosaurs relative to other vertebrates.z = 0.51), and nearly as well on a compliance regime. All synapsids were best adapted to a strength regime, and although sample sizes for non-mammalian synapsids were limited, we did find that pelycosaurs performed poorly, with a large increase in cynodonts and then again in mammals. Given the majority of the vertebrates exhibit a strength regime, we hypothesize that SE/stiffness evolved via an OU-like process of optimization, whereas MA and AR evolution is better explained by a BM process as they were apparently not optimized in most vertebrate groups examined.The landscape of stem tetrapodomorphs does not provide any clarity owing to limited sample size; although they are ‘best’ adapted for the strength regime, they perform poorly on it ( (g)By contrast to the hypothesized evolutionary modes based on adaptive landscape analyses, the best-fitted trait evolution models for all biomechanical traits  across vertebrates are OU models. An OU model is also a better fit than either BM or ACDC when mammal and non-mammal datasets are fitted separately. There is no consistent evidence for evolutionary rate change in biomechanical traits in mammals versus non-mammals. In 68% of trees, laurasiatheres  or all crown Mammalia are reconstructed with substantially lower rates of evolution in MA compared with other jawed vertebrates. No clade exhibits substantial increase in MA under a 50% majority rule criterion. In 74% of trees New World monkeys/primates exhibit a two-order increase in AR rate evolution. In 69% of trees Toxicofera  or Serpentes exhibit greater than 80% reduction in AR evolutionary rate. In jaw stiffness, 93% of trees show a substantial slowdown (less than 10% of average rate) in Euarchontoglires  and a substantial speed-up (5–10 times average rate) for ornithodirans (dinosaurs and pterosaurs). (h)cited1, gbx2, dlx4, meis2 and rgs5 exhibit the largest differences from other jaw development genes sampled . By contrast, dlx2 shared similar relative taxonomic differences to most other genes sampled. The default set of relative evolutionary rate analyses used no transformations, weighting or scaling. The results show that evolutionary rates in four genes  are significantly (padjusted < 0.05) correlated to jaw AR evolution. Only dlx1 is significantly correlated to MA evolutionary rate. Evolutionary rates of three of the analysed genes  are significantly correlated to jaw stiffness evolutionary rate (a). A series of sensitivity analyses were conducted using other combinations of transforms, weights and scales; these analyses provided differential support for the significant correlations identified above .The calculated entanglement values suggest that the genes ary rate a. A serialx3, whereas euarchontoglires exhibit an ancestrally accelerated plagl1 evolutionary rate. Both clades are also reconstructed to have relatively low rates of bmp3 evolutionary rate and no ancestral change in rgs5 evolutionary rate. No other clade exhibited statistically significant deviations from the overall evolutionary rates. The genes found to be significantly correlated with biomechanical trait evolutionary rates show the same relative contribution to genotype–phenotype correlation in the path analysis (b). There is a significant but weak correlation between phenotype and biomechanics, with stiffness (maximum deflection) contributing the most to this association (path coefficient = 0.59), followed by AR (0.08) and MA (0.02).Relative evolutionary rates of the analysed genes were plotted over the composite phylogeny , and laurasiatheres are reconstructed as having ancestrally accelerated evolutionary rate for analysis b. There . 4Functional morphological analyses of the vertebrate lower jaw indicate the presence of biomechanical decoupling and trade-off associated with the morphological diversification of the mammalian single-element mandible. Mammalian and non-mammalian jaws are distinguished by a switch in morphological and biomechanical trait disparity relationships. Bootstrapped and rarefied disparity metrics show statistically significant increases in mandible shape disparity in mammals . On the other hand, mammalian jaw MA, AR and stiffness were significantly lower in disparity, even after accounting for uneven taxon sampling. Furthermore, mammals exhibit significantly higher jaw stiffness, but lower jaw efficiency (MA) and speed (high AR/low jaw velocity), compared with non-mammals . This coe,f). However, the lower biomechanical trait disparity associated with the mammalian mandible within this strength regime reinforces the presence of functional constraints despite high shape disparity and stiffness; many differently shaped mammalian jaws exhibit similar mechanical traits. Furthermore, the significant increase in jaw stiffness comes at the expense of lowered jaw efficiency and speed . Mammals and non-mammalian synapsids maintain this strength regime , but sauropsids  have an optimized adaptive landscape characterized by an AR regime focused more on compliant and faster-closing jaws . During their evolution, crown mammals, with their single-element jaw bones, optimized on the existing strength regime within the adaptive landscape by further elevating jaw stiffness e,f. Howend speed . Taken tGiven the findings of the adaptive landscape analyses, we hypothesized that speed (AR) and efficiency (MA) functions evolved without evolutionary rate shifts during the transition from mammals to non-mammals; we also predicted that jaw stiffness experienced an evolutionary rate shift between mammals and non-mammals. We tested these hypotheses by modelling AR, MA and stiffness trait evolution using a variable rate shift model and found no evidence for evolutionary rate shifts in AR or MA at the transition to mammals. We also did not observe a rate shift in stiffness in the transition to mammals, although the fitted model suggests Euarchontoglires  experienced a substantial slowdown in stiffness evolutionary rate, in part reflecting the decreased disparity in stiffness values. These findings indicate that the OU-type jaw stiffness evolution suggested by the adaptive landscape analysis and supported by trait evolution modelling did not couple with modified evolutionary rates of change in jaw stiffness in all mammals; rather, the increase in jaw shape disparity in mammals occurred without modifying the jaw stiffness evolutionary tempo or mode already observed in their non-mammalian relatives. Furthermore, although adaptive landscape analyses optimized stiffness/strength as the primary regime and stiffness data best fit an OU process of evolution, both AR and MA trait evolution fit an OU model as well; this is interpreted as evidence of a biomechanical trade-off where all three traits may be evolving toward their respective functional optima, but strength/stiffness appears to be the principal trait that mammals and non-sauropsid vertebrates in general optimized at the expense of efficiency and speed. This observation suggests stiffness could be a universally optimized functional trait across vertebrate clades and is evidenced by its importance in mechanically demanding feeding tasks as disparate as suction feeding  and bonea). Stiffness evolutionary rates are significantly correlated with alx3, rgs5 and bmp3. MA (correlated to dlx1) and jaw velocity  trait values are also found to be genetically linked. Nested expression of the dlx gene family by cranial neural crest cells is associated with normal mandible development and jaw adductor formation, and is also known to regulate the downstream expression of plagl1, alx3, rgs5 and other genes during normal jaw development [Relative gene–biomechanics evolutionary rate analyses demonstrate that the narrower range of biomechanical trait values that distinguish mammalian jaws from non-mammalian jaws is genetically correlated a. Stiffnelopment . The asselopment ,59. Giveb; path coefficient = 0.01). Among the biomechanical traits, SE explains the most variance in the jaw shape–biomechanics correlation among the three measured traits (SE path coefficient = 0.591 compared with 0.084 for AR and 0.019 for MA). These findings suggest that the low disparity of jaw stiffness in mammals is in part related to correlated rates of biomechanical evolution with jaw development genes, but such correlation is only weakly associated with overall jaw shape. We speculate that genetically encoded structure–function linkages for jaw stiffness are localized in certain regions of the jaw rather than to the overall shape. Future comparative evolutionary developmental studies would provide insights into the expression patterns of these biomechanically relevant candidate genes.Path analyses show that the genes with the highest degree of correlated evolutionary rates to biomechanical variables also contribute the highest amount of variance towards the gene–jaw shape covariance, which is statistically significant but weak (figure 4b). Such a pattern of high shape disparity coupled with lower biomechanical disparity explains previous findings of redundant and complex form–function mapping of mammalian feeding systems at lower taxonomic rank levels [The presence of many-to-one form–function mapping in the mammalian mandible suggests that the single-element lower jaw may be an example of a ‘fly in a tube’ model of evolution, whereby potential for evolutionary disparity and/or rate shifts within a trait complex  is constrained in certain regions of a morphospace and shaped by genotypic and/or phenotypic integration . The absfigure 4k levels  and suggk levels ; this phk levels .Figure Body size is an important organismal trait underlying many biological scaling relationships ; in the Although it is likely that functional morphological variation in 3D bone material properties and shape are not fully captured by the 2D mandibular profiles examined in this study, the significantly non-random distributions of both shape and biomechanical traits in our dataset suggest critical functional signals are still present in 2D data. It is likely that MA, AR and stiffness measures taken from 2D jaw shapes only partially capture the biomechanical characteristics of 3D jaw function, but any systematic errors in estimating those traits using 2D data should not bias the interpretations made herein, unless there are significant differences in how well 2D represent 3D structures from clade to clade. On the other hand, the extent to which jaw shapes of different vertebrate clades overlap each other in morphospace may change if 3D shapes are used; this may be particularly important for taxa in which there are substantial mediolateral morphological and biomechanical differences. Despite these potential shortcomings, the important role of dorsoventral kinetics and kinematics of mammalian and all gnathostome vertebrate jaws , which increased in MA relative to the movement about a mediolateral axis in crown mammals ,68. ReseAlthough mandibular evolution was once thought principally to have enabled mammals to innovate in feeding mechanical versatility and efficiency over those of non-mammal vertebrates, the new data presented here across a wide sample of vertebrates clearly document that stereotyped stiffness in the mandible was a pre-existing condition underpinning the diverse jaw shapes expressed in crown mammals. A further optimized stiffness regime across diverse mammal jaw shapes evolved at the expense of efficiency and speed, but such a regime may have served as the hotbed for the radiation of dental morphologies and masticatory strategies that exemplify mammalian biodiversity. These new findings should encourage further investigations into how crown mammals radiated into new adaptive zones and became ecologically dominant in various ecosystems during the Cenozoic by capitalizing on a fundamentally modified and stereotyped jaw form–function configuration.https://doi.org/10.6084/m9.figshare.c.6484657 [The data is available at: .6484657 .


Rice is a staple food and energy source for half the world’s population. Due to its quick digestion and absorption in the gastrointestinal tract, rice is typically regarded as having a high or medium–high glycemic index (GI); however, this can vary depending on the variety, nutrient compositions, processing, and accompanying factors. This report included a table of the glycemic index for rice and rice products in different countries, which could give an overview and fundamental information on the recent GI of different rice varieties. In addition, latest updates about the mechanism effects of rice nutritional profiles and processing techniques on GI were also provided and discussed. The influence of state-of-the-art GI regulation methods was also evaluated. Furthermore, the effectiveness and efficiency of applied technologies were also given. Furthermore, this review offered some aspects about the potential nutraceutical application of rice that food scientists, producers, or consumers might consider. Diverse types of rice are grown under various conditions that could affect the GI of the product. The instinct nutrients in rice could show different effects on the digestion rate of its product. It also revealed that the rice product’s digestibility is process-dependent. The postprandial glucose response of the rice products could be changed by modifying processing techniques, which might produce the new less-digestive compound or the inhibition factor in the starch hydrolysis process. Because of the significant importance of rice, this paper also concluded the challenges, as well as some important aspects for future research. However, depending on the type of product, the analytical method is also different or adjusted to suit actual conditions.According to the World Health Organization, at least 347 million people are suffering from diabetes. And this number is increasing day by day; it is predicted that by 2030, this number could reach double the current number . DiabeteOryza sativa L.), which contains 60–80% starch depending on the variety [A staple food for almost 4 billion people worldwide, rice supplies 15% of protein and 21% of energy per person globally . In Sout variety ,10. Howe variety . The nut variety . However variety ,12, but  variety . HoweverIn recent years, many methods of reducing the GI of rice have been extensively studied, which can improve on previously developed methods or newly developed methods. Parboiling, germination, and polishing with various conditions were commonly applied to modulate the GI of rice grain ,15,16,17As a natural form of carbohydrates, starch is one of the main sources of energy in the human diet. Chemically, amylose and amylopectin are the two forms of polyglucans that form starch. Amylose typically takes the form of a linear polysaccharide made up of D-glucose units connected by α-(1-4)-glycosidic linkages. While amylopectin is considerably larger than amylose and extensively branched, it also has α--glycosidic branch points in addition to α-(1-4)-glycosidic linkages .In the human body, disaccharides are first formed by the hydrolysis of starch by salivary and pancreatic amylase enzymes in the mouth and small intestine, respectively. The first product of this process is usually maltose and a small amount of dextrin, which is further broken down to glucose by the two enzymes maltase-glucoamylase and sucrase-isomaltase at the brush border . The endThe GI is defined as the incremental blood glucose area following the test food, expressed as the percentage of the corresponding area following a carbohydrate equivalent load of a reference product” [The glycemic index (GI) is a method used to classify dietary carbohydrates based on their impact on the blood glucose response after intake of starch- or sugar-rich meals. It was first introduced by Jenkins et al. as follows: “product” . Accordiproduct” . Moreoveproduct”  previousDifferent in vitro protocols were applied for the determination of the GI of rice and rice products, which mainly depended on the purpose and goal of the studies. The method of Goñi et al.  was widein vivo parameters of the human gastrointestinal tract. It offers a quick and simple standardized method to analyze how food or pharmaceuticals behave during the digestion process. Utilizing this technology would result in less variance in the composition and structure of the food matrix than other methods, which would be advantageous [Different digestion conditions, such as enzyme activity, incubation durations, and the change in pH, were considered in several alternate protocols as shown in ntageous . Furtherntageous  reportedThe comparison of static and dynamic in vitro protocols for starch hydrolysis of rice pudding was also recently published by Li et al. , which iin vivo, which requires the use of animal or human subjects for evaluation of the two-hour blood glucose response after food intake. When investigated in vivo, the digestive process is better understood. However, this method has difficulties in terms of ethical considerations, intricacy, and tediousness [The GI of rice product is also determined iousness . Besidesiousness . A GL abiousness . The glyiousness .Regardless of time and money consumption, for animal testing, six-week-old Wister male rats, under formal testing ethical approval, were recently selected to measure the glycemic index of rice in Bangladesh . At 0, 3in vivo glycemic response of cooked parboiled rice, took the blood and measured the blood glucose level from 25 heathy volunteers (BMI = 18.5–23 kg/m2) after consuming standard and rice sample (0–120 min) on a separate day. The proportion of food that they consumed contained 50 g available carbohydrate. Fasting is required for the subjects and Human Ethics should be approved before testing. Pre-diabetic subjects were also considered to choose the appropriate rice for health promotion [The glycemic index was also measured in the human body. The early recommended method of the FAO/WHO Joint Expert Consultation  was applromotion .in vivo tests are summarized in Rice is the second most extensively consumed cereal globally, following wheat. Rice serves as the primary dietary staple for over two-thirds of the global population. In Asia, a population exceeding 2 billion individuals relies on rice as their primary source of nutrition, constituting 80% of their energy requirements. Rice is composed of approximately 80% carbs, 7–8% protein, 3% fat, and 3% fiber. However, the percentage of carbohydrate, which is mainly related to the value of GI, differs by variety and geography . For exaThe most important source of calories for communities around the world is carbohydrates, which have been divided into groups based on how easily they can be digested and how they behave in the digestive system. The overall discussion in this section is outlined in Rice was divided into three groups, including classification as waxy (0–2% amylose), very low amylose content (2–12%), low amylose content (12–20%), intermediate amylose content (20–25%), and medium-to-high amylose content 20–33%), based on the ratio of amylose and amylopectin, which are known as the two main compositions of starch 3%, based. Recent Restricted gut enzyme access to starch is caused by the formation of complexes between amylose and lipids under various heat-treatment methods ,20,40. AThe structure of polysaccharides also affected the ability of resistant starch to the hydrolysis of amylolytic enzymes. The short chain of amylose could promote the reassociation and re-order process to form the new compound which is intolerant of the hydrolysis enzyme . It is wThe other two main compositions are protein and lipid, which also greatly affect the digestion rate of rice and rice products. The protein content of rice grain/flour ranged from 4.5% to 15.9%, while the lipid content was much lower, as shown in Lipids and amylose have long been understood to produce inclusion compounds, with the hydrocarbon part of the lipid residing inside the helical cavity of amylose. These complexes’ slow digestion has been linked to decreased postprandial insulin and blood glucose levels . Firstlyvan der Waals forces, lead to a reduction in the hydrolysis rate of rice starch [in vivo. In addition, the most interesting, in recent years, is colored rice, known as pigmented rice, which is also considered to have a promisingly low GI. When comparing the GI of white rice and colored rice, due to the high number of antioxidant compounds as endogenous phenolic compounds, the observed GI and GL were lower in pigmented rice [In recent years, there has been increasing interest in the effects of various polyphenols on the in vitro digestibility of rice starch, rice flour, and rice products. Polyphenols could not only act as inhibitors of enzyme hydrolysis but could also interact with starch to form starch–polyphenols complexes ,63,64. Re starch . Furthere starch , which pe starch . A high e starch . Recentle starch  also shoted rice . Riceberted rice . The colted rice . TherefoAnother compound that affects the glycemic index of rice, and its product is dietary fiber. Dietary fiber (DF), principally the non-starch polysaccharides of the plant cell wall, is an important component of our diet . DF is aQuality characteristics and the glycemic index of rice could enhance and control by the parboiling process, which is known as a hydrothermal process. The parboiling process led to starch swelling and gelatinizing, the denaturation of the protein structure, and the formation of the lipid–amylose complex in rice . The maiThe production of graded and polished white rice from harvested paddy is a part of post-harvest paddy processing, which was additionally known as dehulling. Dehulling involves separating several components, including brown rice, hull, and dehulled rice. The dehulled paddy is subsequently milled or polished to produce raw white rice (head rice and broken). The ideal milling operation will yield varied amounts of hull (20%), bran (8–12%), milled rice, or white rice (68–72%), depending on the type of rice and the level of processing. For later usage, this is then packaged and stored. Rice is usually eaten as milled grains that have had the bran layers  removed. This type of rice is known as white or polished rice. This polished rice has superior cooking and eating qualities compared to brown rice . The ricIn brown rice, the non-starchy polysaccharides that make up the bran and cell wall of the endosperm serve as a barrier to the starchy endosperm. Any instability to the intact bran layer, which is removed during polishing, can enhance the rate of starch digestibility because digestive enzymes can more easily access the starchy content . VariousFor human consumption, rice is cooked. During that process, the starch of the grain gelatinizes, giving the finished product desirable textural qualities. Additionally, cooking can increase the bioavailability of many nutrients. Different methods were applied to cooking rice as conventional methods, including pressure cooking, cooking in electric cookers, or using microwave or stir-frying, which led to differences in the quality and characteristics of cooked rice . In genein vivo studies [Flour milling, known as the size-reduction process, is a crucial step to receive the fine flour for applying in food products, such as bread, cake, cookies, etc. The particle size distribution in rice flour has greatly influenced the physicochemical properties and the digestion rate of rice flour in both in vitro and  studies ,86. The  studies  examinedIn general, rice starch was extracted from its flour. The most notable difference between rice starch and rice flour is the contents of protein and lipid. Industrial products and functional foods frequently contain rice starch. Starch is mostly separated from protein, fiber, and fat during the isolation process. Thus, it is crucial to avoid amylolytic or mechanical damage to the starch granules, deproteinize the starch effectively, minimize the loss of tiny granules, and prevent starch gelatinization . The alkRice value-added products and processed rice are the most important parts in the food industry. A recent report showed that 10% of the total amount of rice available is turned into various value-added products, in addition to being consumed as whole rice . In thisExtrusion technology was applied to produce various rice products, such as modified starch-based snacks, precooked breakfast cereals, snacks developed from a variety of cereal blends, vegetable-based foods, etc. The main step of extrusion process concludes mixing, heating, cooking, sharing, and shaping, which have an effect on the digestion rate of the product ,88. RiceOne of the low-cost techniques for preparing rice that can enhance its nutritional value is germination. Although brown rice has a relatively high nutritional content, it is not commonly consumed since it is challenging to prepare and indigest and has poor textural qualities in addition to difficulties with long-term preservation . GABA, pA significant amount of dietary carbohydrates and energy is found in cereals, such as rice, which is consumed as a staple food by a vast population worldwide. The glycemic index of rice and rice products may also be categorized as medium–high. In most cases, rice is consumed with other food items in steamed or boiled form . Due to the increased protein, fiber, and fat from other foods in the mixed meal, it is expected that the GI of rice would differ from that of only rice consumed. Recently, some research showed the effects of the mixed meal on postprandial hyperglycemia . FortifiRetrogradation is a phenomenon of starch gel. Following cooling and gelatinization, the molecules of amylose and amylopectin reassemble to produce a more organized structure. Due to the nutritional importance of this process, which involves the release of glucose into the bloodstream gradually from retrograded starch, it is less susceptible to enzymatic digestion . SeveralRice is a staple food in most Asian countries, which is also considered as a main material in numerous products, such as rice cakes, rice noodles, prepared breakfast porridge, and baby food. Because of the adverse effects of high-GI foods, the modification method was developed to fulfill consumer requirements. However, depending on the materials or food products, the method and effectiveness of the technique are different.Recently, most studies have shown that brown rice or colored rice have a lower glycemic index than white rice due to its nutritional profiles and functional properties. The degree of polishing and milling could vary the glycemic index of rice. For instance, the GI of Sinandomeng brown rice (Philippines) was lesser, by approximately 26%, than milled rice . AnotherVarious methods were also applied to reduce the glycemic index of raw rice (uncooked form) as shown in Given the popularity of cooked white rice as a staple diet, finding the methods to reduce its starch digestibility is a key point for the diabetic population. Recently, Shen et al.  found thRice flour  is a form of flour made from finely milled rice, while the rice starch was extracted from rice flour using the common method, also known as alkaline extraction. The endogenous compositions of rice flour/starch made up their GI, which is discussed in Nowadays, physical modification is the trending method due to growing consumer health issues. Physical treatments are not only considered chemical-free methods, but they also show effectiveness in reducing the GI of rice flour/starch . InnovatRice can produce a variety of products, such as porridge, bread, noodles, or cakes. In recent years, scientific researchers have also been developing recipes, as well as suitable preservation and processing methods to create products with high sensory quality that can reduce the GI of the product. By various combinations of different ingredients, as well as the appropriate operation method, low-to-medium GI products have been created, as shown in in vivo tests also needs to be provided to better understand the effect on human metabolism. Additionally, the addition of different ingredients to the formula for rice products could lead to a variation in the digestion rate. To establish a larger study database for upcoming efforts on decreasing glycemic effects, it is possible to conduct scientific studies on these components and foods. Additionally, the technologies used need to be optimized to ensure economy while still creating quality products, which is the main consideration of food scientists and the industry.The report of the International Diabetes Federation stated the following: “537 million adults (20–79 years) are living with diabetes—1 in 10. This number is predicted to rise to 643 million by 2030 and 783 million by 2045” [