Challenges for the food/non-food applications of starch mostly arise from its low stability against severe processing conditions , inordinate retrogradability, as well as restricted applicability. These drawbacks have been addressed through the modification of starch. The escalating awareness of individuals toward the presumptive side effects of chemical modification approaches has engrossed the attention of scientists to the development of physical modification procedures. In this regard, starch treatment via ionizing  and non-ionizing  radiations has been introduced as a potent physical strategy offering new outstanding attributes to the modified product. Ionizing radiations, through dose-dependent pathways, are able to provoke depolymerization or cross-linking/grafting reactions to the starch medium. While non-ionizing radiations could modify the starch attributes by changing the morphology/architecture of granules and inducing reorientation/rearrangement in the molecular order of starch amorphous/crystalline fractions. Apart from its naturally occurring presence in foods, starch has been involved in various food/pharma formulations to endow novel promising characteristics to the fabricated products. However, the widespread application of starch has been restricted by some limitations including its low shear/thermal resistibility, as well as retrogradability. Presently, starch modification through chemical  physical  and enzymatic techniques has been the most developed approach to come across such shortcomings . Apart fRecent years have witnessed the increasing focus of researchers on developing promising modified starches through their exposure to ionizing/non-ionizing radiations. More interestingly the emergence of new starches extracted from new sources has drawn the attention of scientists to explore their behavior under various irradiation conditions. Thereby, the main focus of this review was the recent advances in the impacts of ionizing/non-ionizing radiations on various features  of starches extracted from conventional and novel sources.22.160Co) and cesium-137 (137Cs). On the contrary, electron beam consists of accelerated electrons, which are charged particles produced by regular electricity using usually linear accelerators that do not involve sources of radioactive isotopes (The most applied ionizing radiation types to process/modify biomacromolecules are gamma rays (electromagnetic radiation) and electron beam (high-energy particle radiation). Gamma rays are emitted by radionuclides such as isotopes of cobalt-60  Macromolecule Factors- type of macromolecule, water content, density, dielectric properties, penetration depth, temperature, and ii) Radiation Processing parameters - radiation type, radiation source, process variables , system geometry, oxygen presence, exposure time.3Numerous researches have explored the use of ionizing radiations to prevent food microbial deterioration without compromising the food safety, quality, or nutritional content . However, the molecular structure and composition of their constituents, particularly those of starch, proteins, vitamins, and lipids, could be altered upon irradiation. For instance, a). They suggested that α-1,6-glycosidic linkages are more prone to breakage than α-1,4-glycosidic bonds resulting in the formation of short chain amylose-like structures. In another study the molecular structure of EB treated rice starch was assessed by  radiation on starch molecules. In a recent attempt  studied Fig. 2essed by . Their Hessed by  outlined3.23.2.1c-f). Comparable morphological changes were obtained in case of gamma irradiated broad bean starch  , moisture content of the specimen and the crystalline pattern of the starch are the dominant factors determining the morphological behavior of starch upon irradiation. In a recent attempt  revealedFig. 2n starch , bean stn starch , lotus sn starch , red potg. 2g-j)  and lotug. 2g-j) . On the g. 2g-j)  that stug. 2g-j) , or the g. 2g-j)  report wb). .  also obs3.2.2k) which could be the result of its more sensitivity against irradiation.Starches with different crsytalline pattern have experienced various alterations upon irradition. In general, without any change in the crystalline pattern of the starch, its relative crystallinity would be reduced following irradiation which causes a drop in intensity of X-ray diffraction peaks. This change could be the result of the disordered starch granule’s double helices and the destruction of the ordered structure (i.e. amylopectin). For instance,  obtainedFig. 23.2.3l)  . Similarl) . On the l) , (Navaf l) , and   obtained3.2.4When exposed to ionizing radiations up to 20 kGy, the starches' water absorption capacity (WAC) increases. The structural unwinding of starch branches and the development of simple sugars with a stronger affinity for water are considered to be the causes of the increased WAC. Because it affects various functional and sensory aspects of the food, water absorption is essential to the food preparation process . The swe3.2.5In general, the structural breakdown of starch molecules caused by irradiation, lowers the pasting characteristics , and rheological features . The setback viscosity values provide details on starch retrogradation and syneresis during thawing and storage. The primary chemical that causes greater levels of retrogradation and ultimate viscosity is amylose. Pasting characteristics can be simply linked to the textural characteristics and the final product quality; peak viscosity provides an idea of the capacity to bind water, while a higher breakdown value indicates that the final product will taste better. These characteristics are quite significant and helpful when developing the processing parameters (44.1Microwaves are non-ionizing radiation that has frequencies between infrared and radio waves (300 MHz-30 GHz). Microwave is electromagnetic waves of radiant energy . During 4.1.14.1.1.1Microwave radiation modifies the physicochemical characteristics of starch by affecting water evaporation, swelling power, crude protein, and solubility. These characteristics can vary depending on the type of starch, moisture content, processing time, temperature, and absorbed energy. Numerous investigations have employed microwave treatment to modify starch characteristics. It has been reported that microwave could reduce moisture content, crude protein, crude fiber, water absorption, sugar level and absolute density while it increased the ash level . HoweverIn the case of starch, the molecular rearrangements, which alter the hydration and pasting properties, require a little amount of water in order to absorb radiation and to have a plasticizing impact. Thus, the moisture content is one of the main factors that influences the extent of alterations in presence of irradiation. In an attempt, the ability of MW to alter the techno-functional characteristics and gel viscoelasticity of starchy flour system was investigated by MW irradiation may cause a break in α-1,4 and α-1,6 glycosidic linkages. Thus, the amylose and amylopectin content could be affected upon MW. It has been found that the amylose content of MW treated starches is significantly higher than the native starch, and this can be predicted by the disintegration of amylopectin branches and its long-chain structure. Moreover, the amylose that was held inside the granule structure by the amylopectin groups can escape the semi-crystalline structure due to the swelling of starch during MW and contribute to the measured amylose content . It has 4.1.1.21HNMR spectroscopy further supported these conclusions  are less hampered by the steric hindrance and are more mobile than those located in the crystalline lamellae, they appeared to be more vulnerable to MW energy, which is the likely the source of a selective degradation. It has been stated that the central amorphous region is more susceptible to degradation. The major degradation occurs in the internal chains at the first stage (5 min), while the external chains  mostly destroy at the second stage (10 min) as stated by . They alclusions . This ficlusions .Table 2A4.1.1.3c)   and potato  starches.The morphological properties of starch are mostly dependent on the amount of applied MW treatment where some of the starch granules can retain their structure when treatment is below the gelatinization temperature while, beyond the gelatinization temperature the granular structure could be completely destroyed . During Fig. 3c) . PinholeFig. 3d) . The surFig. 3d) . The sagFig. 3d) . Similar4.1.1.4k). The swelling power values of waxy and non-waxy hull-less barley kernels reduced with the increasing MW conditions (power × time). The internal structure of starch granules varies as a result of MW radiation, which may result in a rearrangement in the crystalline areas and cause variations in starch swelling   , and milk) .4.2k). They also indicated although MW induced no change in starch crystallinity, RF diminished the RC of modified starches  and concluded that RF could be a potent technique for annealing modification of starches. The impact of RF on physicochemical and structural attributes of starch are provided in detail in Apart from microwave, other types of non-ionizing radiations viz. radiofrequency radiation, ultraviolet radiation as well as infrared radiation could also provoke various alterations to physicochemical, structural and nutritional attributes of starches. However, compared with microwave, limited studies have investigated the impact of these methods on starch characteristics. Radiofrequency radiations are electromagnetic waves possessing the longest wavelengths and smallest frequencies (1––300 MHz) which induce severe molecular rotation/frictions within the medium. Although the principle of generating heat upon RF is similar to the microwave process, the product processed by RF possesses superior quality. Given the longer wavelength of RF waves, they are more potent to penetrate deep into the medium thereby distributing heat more uniformly throughout the product. Hitherto, the successful application of RF in food processing has been reported by numerous researchers . RF treaFig. 3Ultraviolet radiation is another type of electromagnetic radiation with wavelengths ranging from 100 to 400 nm. UV through indirect photochemical reactions, such as the generation of free radicals, might cause ultrastructural alterations. Reportedly, the surface, hydrophilicity, and thermal stability of polysaccharides can be altered upon UV radiation. Due to the poor penetration of UV radiation, rheological and textural changes could occur only at the surface of UV irradiated materials . In this5Starch-based materials have received great attention in the agricultural, food, packaging, and biomedical sectors owing to their biodegradability and low cost . Applyin5.1Besides being cheap and efficient, the packing industry understands that synthetic polymer-based packaging are not biodegradable and cause environmental damage. For this reason, studies on starch and starch composites have been initiated in recent years to elaborate starch based films and packings. Normally, starch in its native form is not suitable for such applications, but when modified, can be employed alone or in blends/composites to improve the barrier properties and thermal and mechanical characteristics of the films . Further−1, and compared with native starch. The applied doses increased the tensile strength of the films, while the water vapor permeability decreased with the increasing doses. According to the authors, between all treatments, the 30-kGy irradiated corn starch film showed great potential for developing biodegradable starch film with improved properties.The commonly used biopolymers are starch, chitosan, alginate, gelatin, and shellac, but poor mechanical properties and hydrophilic nature are the major drawbacks of the films made with these materials. For this reason, numerous studies are in progress to overcome these limitations to offer physicochemical characteristics comparable to those of synthetic polymer-based materials ; within Another study conducted by 5.2Hydrogels are formed by physically or chemically crosslinked macromolecules, forming a three-dimensional network capable of retaining water without disintegration . Their eIn another attempt, The effect of degree of substitution and irradiation on the properties of hydrogels prepared from carboxymethyl-sago starch and polyethylene glycol were investigated by 6The digestibility of foods determines how effectively the gastrointestinal tract digests and absorbs the macronutrient . The molThere are also numerous attempts investigating the impact of non-ionizing radiation on starch digestibility. In this line,  revealed7There has been an increasing interest towards the development of starches with modified morphologies and functionalities offering advantages over their conventional counterparts in various food and biomedical applications. Although ionizing/non-ionizing radiation treatments are proven to be rapid and effective techniques for improvement of nutritional and techno-functional properties of starches, there are still some issues that need to be addressed. The relatively high cost of radiation-assisted modification compared to the commonly used conventional techniques and the limited availability of the irradiation facilities contribute to the failure of their widespread industrial applications. In case of ionizing irradiation, the main limitation of irradiation treatment in food industrial applications is the toxicological hazards it imparts to food materials. In this regard various national and international agencies has been established to develop strict legislations for irradiated foods . Moreover, functional properties of starches treated by radiation are shown to be affected by the radiation type and treatment conditions applied. Besides, bioactivity of special interest such as antioxidant activity are indicated to be negatively affected by the irradiation dose . On the Dr. M.R. Nemțanu acknowledges that her contribution to this work was supported by a grant of the Romanian Ministry of Research, Innovation and Digitalization, CNCS - UEFISCDI, project number PN-III-P4-PCE-2021-1778, within PNCDI III.


Oyster Farming is one of important fisheries and aquaculture industries in Taiwan. Each year, approximately 4000–5000 tons of discarded bamboo scaffolding (BS) used in oyster farming, are generated, so the treatment and utilization of BS should be taken seriously. This study evaluates the suitability of BS for pulp and papermaking by assessing the chemical compositions, microstructural, and fiber morphology. The pulping properties is investigated by soda pulping. The chemical composition of BS shows the potential for application in pulping. The BS microstructure shows that can enhance pulping reactions, while the fiber morphology indicates the possibility of producing high-strength paper. Through the pulping experiment, it demonstrated that BS is suitable for pulping with lower NaOH dosage and longer digestion time. The condition at 170 °C with 14% NaOH dosage for 90 min digestion has the highest yield. After refining the highest pulping yield BS pulp, it can improve the handsheet strength and bulk of the OCC-BS mixed pulp, which can achieve the strength property required for industrial paper. In summary, BS exhibits the potential for pulping application and produces a better paper strength than OCC pulp, exhibiting the feasibility of enhancing the circular utilization value of BS in Taiwan. The floating raft is usually constructed with 43 bamboo poles, including 3 to 4-year-old Thorny, Makino, or Long-branch bamboo of 34 poles with a length of 9 m, and 4-year-old Moso bamboo of nine poles with a length of 8 m. Hence, the rafts are also referred to as bamboo scaffolding. The service life of bamboo scaffolding is approximately two to three years. After its use, the discarded oyster farming bamboo scaffolding (BS) needs to be managed and recycled. Almost 4000 to 5000 tons of BS are generated each year3.Oyster farming is one of the important fisheries and aquaculture industries in Taiwan. The cultivation regions primarily extend along the coastal areas of Hsinchu, Changhua, Yunlin, Chiayi, and Tainan. The methods of the oyster farming include bamboo rafts, hanging, suspension, and long-line methods, with the floating raft culture method being predominantly utilized in the Tainan area5. Additionally, this utilization can be referred to as the utilization of agricultural surplus materials. Nowadays, BS can be utilized in producing biocarbon and wood vinegar, but the quantity of production cannot cover the recycled amounts. Part of BS is breaking down to manufacture the fuel rods by specific company. However, during the processing, the government needs to spend USD 158.04 to 189.65 per ton annually2. Therefore, after the recycling of BS, there is still a need to develop more economically viable and suitable methods of circular utilization. Ho and Lin6 analyzed the chemical compositions of BS, and the result shows a holocellulose content of 75.06%, α-cellulose content of 49.40%, and lignin content of 26.31% in BS, which is similar to native bamboo. In this case, BS may have a potential to be developed as raw materials for value-added fiber applications, thus enhancing its economic value.In recent years, the circular utilization of agricultural waste has gained increasing attention. It can serve as raw material for various purposes, producing high-value-added products7, indicating a growing need for feedstock in papermaking, which becomes imperative to explore novel raw materials in response to the increasing demands in production. In Taiwan, industrial paper is the primary product of the papermaking industry, with its production accounting for 82.0% of the total in 2021, which is also increasing annually8. According to the Taiwanese papermaking industry situation, the recycled paper utilization ratio is 97.9%, the primary feedstock is the old corrugated cardboard (OCC) pulp, but 34.2% of this material is imported. The increasing production exhibits the growing demand for fiber feedstocks, which means the agricultural surplus fiber materials have an opportunity to be applied in industrial paper production according to the Taiwanese papermaking industry trend, and the development of novel materials may have a chance to reduce dependence on imported materials of the Taiwanese papermaking industry. The agricultural surplus fiber materials have been studied and applicated a lot in pulping and papermaking, like rice straw or wheat straw pulping10, but the surplus fiber materials of aquaculture or fisheries, like the wooden boat, bamboo raft, bamboo scaffolding for fisheries, are seldom discussed. These surplus materials still have a circularly utilized potential in fibrous value-added applications.As the paper industry advances, the global demand for paper and paperboard is progressively increasing each year11 conducted pulping of 0.2, 1.2, and 3-year-old Thorny bamboo using kraft and soda methods. For kraft pulping, active alkali (AA) dosage of 16%, 18%, and 20% were used, along with a sulfidity of 25% and a digestion time of 90 min. For soda pulping, AA dosage of 16%, 18%, and 20% were used, with a digestion time of 150 min. Both pulping methods maintained a solid-to-liquid ratio of 1/4, a digestion temperature of 160 °C, and a heating-up time of 90 min. Among these, the 3-year-old Thorny bamboo, which shared the same source as BS raw materials, achieved the highest pulp yield of 40.37% under kraft pulping conditions of 20% AA dosage. For soda pulping, the highest pulp yield was 38.70% under conditions of 16% AA dosage. Mohamad Khair and Masrol12 conducted pulping of 3-year-old Dendrocalamus asper using soda methods, with the conditions of 25% of AA dosage, a solid-to-liquid ratio of 1/7, heating-up time of 70 min, digestion temperature of 170℃ for digesting 3 h. After digestion, the pulping yield is 20.88%. Based on the above studies, research exists regarding the application of soda and kraft methods in bamboo chemical pulping, with the typical AA dosage of 14–21% within pulping conditions. Among the two pulping processes, soda pulping of agricultural surplus materials has been industrially adopted in some countries15. Consequently, this research opts for the soda methods to assess the pulping properties of BS.This study aims to utilize BS in pulp and papermaking, not only providing a novel material for the Taiwanese papermaking industry, but also enhancing the value of BS circular utilization. There is currently no relevant research on pulping and papermaking regarding BS materials, but its raw material is bamboo, so the pulping conditions and methods of this study is designed with reference to bamboo pulping. Chang et al.The purpose of this study is to investigate the feasibility of utilizing BS in pulping and papermaking applications, with the goal of enhancing the circular utilized value of BS. The study evaluates the pulping and papermaking potentials of BS by the chemical compositions, microstructural appearance and fiber morphology analysis. Simultaneously, the original materials (4-year-old Moso bamboo and 3-year-old Thorny bamboo) used to construct BS are act as control groups to analyze material properties and explore changes in BS properties due to seawater immersion. The soda pulping experiment for BS employs a factorial experimental design to investigate how digestion time and AA dosage impact BS pulp properties, including pulping yield , pulp freeness, and handsheet physical properties . After the pulping experiments, the sample with the highest pulping yield is blended with TOCC (Taiwan OCC) in various ratios to examine changes in freeness and handsheet properties. This aims to assess the feasibility of applying BS pulp in industrial paper manufacturing, which means the BS pulp needs to achieve the property level of OCC pulp  to have an opportunity to be utilized. The study hopes to develop a novel circular utilization approach for BS.Phyllostachys pubescens) is harvested from Zhushan, Nantou, Taiwan, and dried by air for two weeks before chopping, and the moisture content is 14.54%. 3-year-old Thorny bamboo (TB) (Bumbusa stenostachia Hackel) is provided by National Pingtung University of Science and Technology , it is also dried by air for two weeks before chopping, the moisture content is 17.23%. After chopping, all the materials are selected out of the small chips with a length of 0.5–1 cm, width of 0.5–1 cm, and thickness of 0.3–0.5 cm, prepared for pulping experiments. The TOCC and JOCC pulps are provided by Houli Mill, CHENG LOONG Co., Ltd. , with freeness of 385 mL and 405 mL and pulp consistency of 39.23% and 37.66%, respectively.Discarded oyster farming bamboo scaffolding (BS) which is already been dried and chopped is provided by Tainan City Fishing Harbor and Coastal Fisheries Management Office , with a moisture content of 9.21%. 4-year-old Moso bamboo (MB)  with a high level (+ 1) of 90 min and low level (− 1) of 60 min, and the NaOH dosage (X2) with high level (+ 1) of 18% and low level (− 1) of 14%. In the meantime, there is a middle point (0) with a digestion time of 75 min and a NaOH dosage of 16%. In the pulping process, the additional added water and NaOH dosage are calculated first, and then the additional added water is used to soak BS chips. After soaking, the NaOH is mixed with the chips and placed into the rotary digester . Then, the pulping process begins, following the condition mentioned above. After digestion and cooling, the pulp is washed under tap water using the 200 mesh sieve until the filtered water becomes colorless. When the pulping process is finished, the pulp is placed into a plastic bag in preparation for analysis.The constant conditions for BS soda pulping experiment include the digestion temperature of 170 ℃, the solid-to-liquid ratio of 1:5, the heating rate of 1.5 ℃/min, and BS weight of 500 g . The solid-to-liquid ratio is different from the bamboo pulping The materials are pulverized by the high-speed pulverizing machine , and the particles between 40 and  60 meshes are screened out for chemical composition analysis, including ash (TAPPI T 211 om-22), alcohol-benzene extractives (TAPPI T 204 cm-17), holocellulose (ASTM D1104-56), acid-insoluble lignin (TAPPI T 222 om-21), and α-cellulose (TAPPI T 203 cm-22), each analysis is repeated twice to calculate the average and standard deviation.2, Oxford Instruments, UK) to determine the presence of any metal elements deposited on BS surface from the sea. A comparative analysis is conducted between BS and reference samples MB and TB.The microstructural appearance of BS is observed using a scanning electron microscope . The operating voltage during the observation is set at 15 kV. Simultaneously, surface elemental compositions are assessed through Energy-dispersive X-ray spectroscopy , and recording fiber length (mm), fiber width (μm), cell wall thickness (μm), and coarseness (mg/m). Otherwise, the L/D value (slender ratio) is calculated by the length to width ratio. All the samples are conducted with six times to record the average and standard deviation.The method of fiber morphological analysis is according to Franklin’s methodAfter pulping, the sample is screened using the flat screen  with the slot width of 0.25 mm according to Tappi UM 204. In the unscreened pulp, the material unable to pass through the 0.25 mm sieve is termed Shives, while that passing through and collected by a 200 mesh sieve is referred to as Accepts. The lost material is collectively termed Fines. After screening, Collective Shives and Accepts are measured the total oven-dried weight and determine Shives, Accepts, and Fines ratios using Eqs. , (2), anThe measurement of pulp freeness is conducted using a Canadian standard freeness (CSF) tester , according to TAPPI T 227 om-17. Each sample is tested twice, and the average result is recorded.The accepted fiber appearance is observed using the optical microscope , and during the experiment, the pulp concentration is adjusted to 0.4% to disperse the fibers for specimen preparation. The images of the fibers are captured by the microscope camera  using the MicroCam V5 software .2 to simulate the grammage of cardboard. After the handsheet making, the handsheet is conditioned under 50.0% ± 2.0% RH and 23.0 ± 1.0 °C for at least 4 h according to TAPPI T 402 sp-13 before testing. The grammage and thickness of the handsheet were measured according to TAPPI T 410 om-08 and T 411 om-21, respectively, then the handsheet bulk was calculated. Six handsheets with similar grammage and intact appearance were screened for strength properties testing. The testing items were mainly the paper properties specified by Taiwan CNS standards for cardboard paper, including tensile index (TAPPI T 494 om-01), burst index (TAPPI T 403 om-15), ring crush index (TAPPI T 818 cm-18). The experimental results are recorded by calculating the average value and standard deviation of six test data.After soda pulping, handsheets were prepared for each factorial point in Fig. 17, using Microsoft Excel  to analyze. The result of handsheet properties, BS pulp blend into TOCC, are analyzed using IBM SPSS Statistics 20  for one-way analysis of variance (ANOVA). The Turkey Honestly Significant Difference (HSD) test was employed for multiple comparisons, employing a 95% confidence interval. A p-value < 0.05 indicated statistical significance.The process of factorial design analysis follows the guidelines outlined in 'Design and Analysis of Experiments 7/e' by Montgomery6, this study showcased lower ash content in BS, similar levels of toluene-alcohol extractives and acid-insoluble lignin content, a relatively lower holocellulose content, and a higher α-cellulose content. The composition variation in BS could be attributed to the diversity of bamboo materials constructed, as well as variations in usage conditions and climate, leading to differences in service life and the final compositions.The chemical compositions of BS are shown in Table 11), suggesting that seawater immersion did not significantly increase the ash content. The holocellulose content of BS is similar to native bamboo, softwood, and hardwood. The acid-insoluble lignin content is higher than rice straw, wheat straw, and hardwood, but similar to native bamboo, and lower than softwood. Meanwhile, the α-cellulose content is similar to TB and higher than in other materials. In summary, the chemical composition of BS suggests its potential for application in pulping.The chemical compositions of BS, native bamboo , and other fiber materials used in pulping are presented in Table 18. Therefore, it can be inferred that BS may be more susceptible to penetration compared to native bamboo during pulping.To figure out the effect of seawater immersion on the fibrous structure of BS, SEM is used for the observation of microstructure on BS to compare with MB and TB, SEM image is showed in Fig. 6 revealed the results of EDS analysis, indicating element content of 2.08% Na, 1.49% Si, 3.72% Cl, and 6.76% Ca. These findings suggest a higher content of sea salt elements on the surface. But the ash content (2.12%) in the BS of this study is significantly lower than that in the study (11.19%) by Ho and Lin6 It can be seen that the BS used in this study has a lower degree of salt deposition, and the ash content is similar to the native bamboo, which indicates that BS of this study is suitable for pulping.To validate the lower ash content result in BS, this study employed EDS analysis to examine the surface elemental composition of BS, in order to confirm the presence of salt deposits from seawater on the surface. The analytical results are presented in Fig. 19. The Fw of BS is closed to native bamboo , and lower than softwood and hardwood. The CWT of BS is thicker, but the coarseness is the lowest in Table 20. The L/D value of BS is lower than native bamboo but higher than wood. El Omari et al.21 suggest that fiber with an L/D value > 70 can produce high-strength properties paper. In summary, the fiber morphology of BS has the potential for application in papermaking.The paper properties are influenced by the fiber morphology of the pulp. Therefore, this study evaluates the fiber morphology of BS, and compares with MB and TB, after defibration using Franklin's method, in order to assess the feasibility of utilizing the BS fibers for papermaking. The results are presented in Table 1X2 interaction, the value is -3.940. The shives ratio is significantly affected by all factors, the absolute values from high to low are X2 |-10.265|> X1X2 |1.735|> X1 |-1.715|, which indicates X2 has more impact on shives ratio. The fines ratio is significantly affected by X2, the value is 10.175.The factorial analysis of pulping yield is presented in Table 11, BS soda pulp demonstrates a higher acceptance ratio with lower NaOH dosage, shorter digestion time, and an even higher solid-to-liquid ratio, which can also be inferred as a pulping result of the lower concentration of NaOH solution and exhibiting the easier condition of the of pulping with BS.The factorial analysis charts of pulping yield are shown in Fig. The accepted fibers are observed under the optical microscope, the images with the magnificent of 40× are shown in Fig. 1 and X1X2 interaction have a significant impact on freeness. The factorial analysis chart  and predominantly contributes to the decline in strength properties. None of the samples underwent refining, resulting in a freeness measurement exceeding 700 mL. Consequently, the handsheets displayed relatively diminished strength properties. To evaluate the feasibility of cardboard production, the BS pulp sample with the highest pulping yield  is refined by Hollander beater for 60 min and blended with TOCC to test the freeness and handsheet properties.The factorial analysis of handsheet properties is presented in Table The result of BS pulp blend with TOCC for papermaking experiment is presented in Table The results of BS material properties present the potential for application in pulping and papermaking, including the higher α-cellulose content, the existence of mesoporous pores, and the fiber morphology suitable for papermaking. After the pulping experiment, the pulp properties  are mainly affected by the NaOH dosage. According to the results, the soda pulping condition of BS with lower NaOH dosage (14%) and higher digestion time (90 min) has the highest pulping yield. The BS pulp blended into TOCC can enhance the freeness, handsheet strength, and bulk properties, exhibiting the potential and feasibility of application in industrial paper production. In conclusion, the BS can be utilized as the feedstock of pulping and papermaking, providing a novel material and enhancing the value of BS in circular utilization.


Objective: To investigate the positive rate of chromosomal and monogenic etiologies and pregnancy outcomes in fetuses with hyperechoic kidney, and to provide more information for genetic counseling and prognosis evaluation.Methods: We performed a retrospective analysis of 25 cases of hyperechoic kidney diagnosed prenatal in the Second Affiliated Hospital of Harbin Medical University and Harbin Red Cross Central Hospital (January 2017–December 2022). Furthermore, we conducted a meta-analysis of a series of hyperechoic kidneys (HEK) in the literature to assess the incidence of chromosomal and monogenic etiologies, mortality, and pooled odds ratio (OR) estimates of the association between the incidence of these outcomes and other associated ultrasound abnormalities.Results: 25 fetuses of HEK were enrolled in the cohort study, including 14 with isolated hyperechoic kidney (IHK) and 11 with non-isolated hyperechoic kidney (NIHK). Chromosomal aneuploidies were detected in 4 of 20 patients (20%). The detection rate of pathogenic or suspected pathogenic copy number variations (CNVs) was 29% (4/14) for IHK and 37% (4/11) for NIHK. Whole exome sequencing (WES) was performed in 5 fetuses, and pathogenic genes were detected in all of them. The rate of termination of pregnancy was 56% in HEK. 21 studies including 1,178 fetuses were included in the meta-analysis. No case of abnormal chromosome karyotype or (intrauterine death)IUD was reported in fetuses with IHK. In contrast, the positive rate of karyotype in NIHK was 22% and that in HEK was 20%, with the ORs of 0.28 (95% CI 0.16–0.51) and 0.25, (95% CI 0.14–0.44), respectively. The positive rate of  CMA in IHK was 59% and that in NIHK was 32%, with the ORs of 1.46 (95% CI 1.33–1.62) and 0.48 , respectively. The positive rate of monogenic etiologies in IHK was 31%, with the OR of 0.80 (95% CI 0.25–2.63). In IHK, the termination rate was 21% and neonatal mortality was 13%, with the ORs of 0.26 , 1.72 , and that in NIHK was 63%, 0.15 ; 11%, 0.12 , respectively. The intrauterine mortality in NIHK group was 2%, with the OR of 0.02 . HNF1B variant has the highest incidence (26%) in IHK.Conclusion: The positive rate of karyotype was 20% in HEK and 22% in NIHK. The positive rate of CMA was 32% in NIHK and 59% in IHK. The positive rate of IHK monogenic etiologies was 31%. HNF1B gene variation is the most common cause of IHK. The overall fetal mortality rate of NIHK is significantly higher than that of IHK. The amount of amniotic fluid, kidney size and the degree of corticomedullary differentiation have a great impact on the prognosis, these indicators should be taken into consideration to guide clinical consultation and decision-making. Congenital abnormalities of the kidneys and urinary tracts (CAKUT) are one of the most common fetal structural abnormalities, occurring in about 30%–50% of prenatally diagnosed malformations . CAKUT oHyperechoic kidneys (HEK) are occasionally seen on routine renal ultrasound scan, or with other clinical indications detected on late pregnancy scans, caused by renal abnormalities , such asThe differential diagnosis of fetal isolated hyperechoic kidney includes autosomal dominant and autosomal recessive polycystic kidney disease (ADPKD and ARPKD) respectively, congenital nephrotic syndrome, renal vein thrombosis and obstructive uropathy . In non-Traditional karyotyping, such as G-banding, has been the standard method for detecting various chromosomal abnormalities for decades. With the improvement of prenatal diagnosis technology, more and more prenatal fetal abnormalities are diagnosed not only by ultrasound and chromosome karyotype detection, but also by the maturity of CMA and CNV-seq technology, which has greatly improved the detection rate of chromosomal abnormalities. Different genetic diagnosis strategies have also found more gene abnormalities related to HEK. The study  showed tThe purpose of this cohort study and meta-analysis was to determine the positive rate of chromosomal and monogenic etiologies, perinatal mortality and related factors in IHK and NIHK fetuses, so that clinicians can determine the prognosis of the fetus and provide accurate counseling for parents better.p < 0.05 was considered statistically significant.We reviewed all 3,995 cases of prenatal diagnosis in the Second Affiliated Hospital of Harbin Medical University and Harbin Red Cross Central Hospital (January 2017 to December 2022), of which 251 cases were fetal kidney related abnormalities. Including ultrasound soft indicators, hydronephrosis and renal structural abnormalities. Ultrasound showed that there were 25 cases of fetal hyperechoic kidney, including 14 cases of IHK  and 11 cIn February 2023, were searched by two researchers using combinations of the following keywords: “fetus,” “antenatal,” “prenatal,” fetus*,” “fetal,” “kidney,” “kidney*,” “hyperechoic” and “hyperechoic*.” For all data-bases, the last search was run on 28 February 2023.http://www.prisma-statement.org/) and MOOSE , and China Biomedical Literature Database (CBM) (from 1 January 1990 to 28 February 2023) were searched by two researchers using the Chinese terms for “fetal,” “kidneys,” and “hyperechoic.” Additionally, English language databases PubMed, Embase, Cochrane Library and Web of Science (from 1 January 1945 to 28 February 2023) were searched by two researchers using combinations of the following keywords: “fetus,” “antenatal,” “prenatal,” fetus*,” “fetal,” “kidney,” “kidney*,” “hyperechoic” and “hyperechoic*.” The amount of amniotic fluid was assessed by a semiquantitative method. Diagnostic criteria were based on ultrasonographic assessment of AFV (amniotic fluid volume) or AFI (amniotic fluid index): AFV ≥ 8 cm or AFI ≥ 25 cm for polyhydramnios, and AFV ≤ 2 cm or AFI ≤ 5 cm for oligohydramnios . Kidneysnd MOOSE  guidelinInclusion criteria were studies reporting fetal, neonatal, and infant outcomes. Renal hyperechogenicity was diagnosed on prenatal ultrasound. The primary outcomes were termination of pregnancy (TOP), intrauterine death (IUD) and neonatal death (NND).Screening was performed independently by two investigators based on inclusion and exclusion criteria. Any disagreements were resolved by discussion with a third researcher. The inclusion criteria is that patients with fetal renal hyperechogenicity confirmed by fetal ultrasound in the second or third trimester, including singleton and twin pregnancies.Exclusion criteria were as follows: 1) Reviews and case reports; 2) animal research; 3) There was no further testing or follow-up outcome.Two researchers independently screened the literature, extracted and cross-checked the data. Differences, if any, are resolved through discussion or negotiation with a third party. During literature screening, the title of the paper was read first, and after excluding obviously irrelevant literature, the abstract and full text were further read to determine inclusion. Contact original study authors when necessary for information not identified but important to this study.Data extraction included: 1) Basic information of the included studies: Research title, first author, published journals, etc. 2) the baseline characteristics of the research object and further detection methods; 3) Key elements of risk of bias assessment; 4) Outcome indicators of interest and follow-up prognosis data.The Newcastle-Ottawa scale was used to evaluate the quality of the included literature, according to NOS, each study is judged on three broad perspectives: selection, comparability and results, i.e., the selection of the study groups; the comparability of the groups and the ascertainment of outcome of interest. Assessment of the selection of a study includes the evaluation of the representativeness of the exposed cohort, selection of the non-exposed cohort, ascertainment of exposure and the demonstration that the outcome of interest was not present at the start of study. Assessment of the comparability of the study includes the evaluation of the comparability of cohorts based on the design or analysis. Finally, the ascertainment of the outcome of interest includes the evaluation of the type of the assessment of the outcome of interest, its length and the adequacy of follow up. According to NOS, a study can be awarded a maximum of one star for each numbered item within the Selection and Outcome categories. A maximum of two stars can be given for Comparability . There ap > 0.10 and I2< 50%, there was no statistical heterogeneity among the results of each study, then the fixed effect model was used for meta-analysis. Otherwise, there was statistical heterogeneity among the results of each study, and the sources of heterogeneity were further analyzed. After excluding the influence of obvious clinical heterogeneity, a random effect model was used for meta-analysis. The test level for the meta-analysis was set at α = 0.05. Methods such as subgroup analysis or sensitivity analysis were used to deal with significant clinical heterogeneity, or only descriptive analyses were performed.Review manager 5.4.1 software was used for statistical analysis. The single group rate was used as the effect analysis statistic, and its 95% confidence interval (CI) was given. The I2 was used to quantitatively determine the size of heterogeneity. The fixed effect and random effect models were used for meta-analysis. When PKHD1 gene. The case No. 5 of IHK has a compound heterozygous PKD1 genotype, involving a pathogenic and an uncertain significance variant, while the fetus is also heterozygous for a COL4A3 gene variant responsible of the autosomal dominant form of Alport syndrome. Only 1 case of WES in NIHK group, the results showed that the suspected pathogenic gene CC2D2A was detected, which was related to Meckel-Gruber Syndrome. The follow-up results showed that 14 cases (56%) had termination of pregnancy (TOP), the TOP rate was 43% (6/14) in the IHK group and 73% in the NIHK group. 11 patients survived well, including 1 case with ADPKD, and no neonatal deaths occurred. In the IHK group, Case 1, and Case 4 had less amniotic fluid, Case 4 was diagnosed with PKHD1 and the pregnancy was terminated, and case 1 was born normally. Case 14 had slightly enlarged kidneys and was born normally. In the NIHK group, the pregnancy was terminated in case 4 with oligohydramnios and case 9 with renal enlargement. However, there was no significant difference in the detection rate of abnormal karyotypes and CNV-seq between IHK and NIHK groups , the fetus was terminated because of multiple malformations, as shown in K groups .Retrieving articles according to predefined search terms, among the 311 Chinese and English articles identified, we eliminated 39 duplicate articles, 118 reviews, consensus and case reports, and 115 articles with irrelevant abstract and title, and finally selected the remaining 39 articles. After further research, a total of 21 articles  were incp-value was 0.22 (95% CI 0.14–0.34).In 6 articles , a totalp-value was 0.20 (95% CI 0.12–0.31).In 10 articles , a totalp-value was 0.32 (95% CI 0.22–0.46).In 5 articles , a totalp-value was 0.59 (95% CI 0.57–0.62).A total of 120 patients with IHK were included in the 6 articles , among wA total of 147 cases of HEK fetuses were included in the 6 articles , of whic2 = 74% and subgroup heterogeneity I2 = 48.4%, pooled OR 0.31 (95% CI 0.15–0.62); However, in NIHK, the total heterogeneity I2 = 68%, and the heterogeneity of subgroups I2 = 81.8%, pooled OR 0.54 (95% CI 0.34–0.88)  . In subg34–0.88) . The totpostpartum neonates and children, such as the need for transplantation surgery, dialysis treatment, hypertension and diabetes drug treatment, there were 30 cases of 147 IHK fetuses in 6 studies , I2 = 30% , intrauterine death (IUD), neonatal death (ND), and even child death during long-term follow-up. 15 studies  recorded studies , with th studies , 20 caseI2 = 30% .HNF1B variants diagnosed in 5 articles , I2 = 49%, p-value was 34%; the mortality rate of normal amniotic fluid volume with an OR of 0.08 (95% CI 0.03–0.22), I2 = 24%, p-value was 7%. There was a statistically significant difference in mortality between the two groups. In NIHK fetuses, the neonatal mortality with an OR of 0.17 (95% CI 0.12–0.25), I2 = 2%, p-value was 15%.Sensitivity analyses were performed by individually excluding each included study to determine whether the combined results of the other studies were stable, and look for sources of heterogeneity. Sensitivity analysis was performed for data with >50% heterogeneity, as shown in Fetal hyperechoic kidney (HEK) is a heterogeneous disorder in etiology . As we kBased on the cohort study of hyperechoic kidney cases in recent years, we hope to find out the common etiologies and prognosis of hyperechoic kidney cases, evaluate the incidence and mortality of different causes, the efficacy of different detection methods for hyperechoic kidney cases, and explore the ultrasonic indicators that affect the prognosis of hyperechoic kidney cases. However, in recent years, the sample size in our cohort study was small, and the observable ultrasound findings were limited. Therefore, we included a retrospective cohort based on previous studies to increase the sample size and reduce the bias caused by insufficient samples. In addition, various ultrasound indicators affecting hyperechoic kidney cases, such as amniotic fluid volume, kidney size and CMD, were collected as far as possible. Another reason is that we wanted to verify the consistency with the results of the meta-analysis through a cohort study.In our study, HEK included was classified as isolated and non-isolated and counted separately. In the cohort study, we found 25 cases of renal hyperechoic in recent years, accounting for about 10% of the cases of prenatal diagnosis of renal abnormalities and 0.6% of all cases of prenatal diagnosis of various indications. In IHK group, two fetuses were diagnosed with trisomy 21, which may seem unexpected, but they had different prenatal diagnostic indicators than ultrasound findings alone. Indications for non-invasive prenatal testing (NIPT) in NO. 19113 suggest a high risk of trisomy 21. Ultrasound images showed only bilateral hyperechoic kidneys. The prenatal diagnosis of NO. 19255 was indicative of advanced maternal age, the actual age was 40 years old, and ultrasound indicated bilateral hyperechoic kidneys of the fetus. Based on the indications, only karyotype and CNV-seq were tested in these two cases. However, hyperechoic kidney was found on ultrasound, we included these two cases in the IHK group. As we know, trisomy 21 syndrome may involve various systemic abnormalities, including some soft indicators, and of course may not be detected by ultrasound in the first and second trimester of pregnancy, perhaps as the pregnancy progresses, their ultrasound will show NIHK.de novo. Although the current research shows that de novo variants are more likely to be pathogenic than hereditary variants, there is still no evidence to judge the prognosis of the fetus, and only regular ultrasound examination can be relied on to assess the survival status and prognosis of the fetus. Therefore, most families still choose to continue pregnancy. Because of the uncertainty about fetal outcomes has led a small number of families to refuse parental CNV-seq testing.It is easy to find that the positive rate of CNV-seq in the NIHK group was higher than that in the IHK group. The positive rate of overall HEK fetal karyotype was 20%, and the abnormal detection rate of pathogenic CNVs was 32%, which was significantly higher than the detection rate of karyotype. The NIHK group also had a significantly higher TOP rate than the IHK group, with an overall TOP rate of 56%. However, due to the small sample size in our cohort, Fisher’s exact probability chi-square test showed that there was no significant difference in the positive rate and TOP rate of genetic testing between the IHK group and the NIHK group. Due to the combination of multiple structural abnormalities, the increased possibility of poor fetal prognosis judged by doctors, and the reasons of family members themselves, it is common for NIHK cases to refuse further WES and choose to terminate pregnancy, especially for families who have offspring and do not have the requirement of childbearing again. In the CNV-seq test results, we found that 6 fetuses carried variants of uncertain clinical significance (vous). At present, the correlation of these variants with ultrasonic manifestations and future pathogenicity is unknown. The risk of birth defects in fetuses without CNVs is currently 0.4% , so the PKHD1 gene in the fetus), and the fetal ultrasound of this pregnancy indicated bilateral hyperechoic kidneys of the fetus. Considering the possibility of monogenic etiologies of the fetus, Trio-WES testing was performed. The prenatal diagnosis indication of NO. 210086 was bilateral hyperechoic kidneys and oligohydramnios indicated by fetal ultrasound. Infantile polycystic kidney was considered, and WES examination was conducted to verify the ultrasound diagnosis. The fetal ultrasound of NO. 210323 indicated bilateral hyperechoic kidneys. Due to the gestational week exceeding 25 weeks, and the pregnant woman and her family members refused cordocentesis, considering the possibility of fetal monogenic cause, amniocentesis was performed and amniotic fluid CNV-seq and trio-WES were examined. The indication of NO. 220253 was bilateral hyperechoic kidneys indicated by fetal ultrasound. After communication with the pregnant woman, 10 mL of amniotic fluid was extracted during amniocentesis for preservation, and the chromosome karyotype and CNV-seq detection of amniotic fluid were performed first. However, CNV-seq results showed that no kidney related copy number variation was found except for X-linked ichthyosis pathogenic variation. Therefore, WES testing was performed on the preserved amniotic fluid. The fetal ultrasound of case NO. 170015 indicated multiple malformations, which was considered Meckel-Gruber syndrome. The family requested termination of the pregnancy and WES testing of the fetal cord of the induced labor was performed to verify the ultrasound diagnosis. In all 5 cases of WES, monogenic etiologies that could not be found by karyotypes and CNV-seq were found, especially PKHD1 was more common in renal hyperechogenicity, followed by PKD1 gene. However, WES results of case 4 (NO. 210086) in IHK group showed complex heterozygous variation of PKHD1 gene, both vous, which were maternal and paternal, respectively. This can lead to PKD type 4 with or without polycystic liver disease (OMIM: 263200), although there seems to be some difficulty in making a definitive diagnosis. Therefore, in this case, the fetal ultrasound examination at 29 weeks gestation showed oligohydramnios (less than that at 24 weeks gestation), bilateral hyperechoic kidneys with enlarged volume, and small bladder, which considered infantile polycystic kidney. Although the ultrasound diagnosis was not completely consistent with the genetic diagnosis, the risk of poor prognosis of the fetus was comprehensively assessed, so we agreed with the parents’ decision to terminate the pregnancy based on the ultrasound diagnosis. For the 2 patients with 17q12 deletion syndrome whose CNVs were found, we did not perform WES testing again, but we speculated that HNF1B gene might be involved. These are common causes of hyperechoic kidney found in studies. Although some of our cases survived well within half a year to 1 year after delivery, some of them did not undergo genetic testing, and renal structural deterioration and even renal function decline may occur several years later, so these cases may need longer time follow-up and more frequent renal examination.Trio-WES tests were performed for 5 cases in which a search for monogenic etiologies was deemed necessary by the physician. Case NO. 210040 prenatal diagnosis was indicated by advanced age of the pregnant woman (>35 years old), adverse pregnancy history  and MCDK with 9% and 8%, respectively. There are other diseases that make up a large proportion of IHK, Such as WTX variant, renal tubular disease, nephroblastoma, autosomal dominant CAKUT (AD CAKUT), Type II Ivemark Syndrome, renal agenesis, Beckwith-Wiedemann syndrome (BWS). The good prognosis and disappearance of hyperechogenicity accounted for 23% of the cases.Due to different years, detection methods are limited by testing technology, and some cases are diagnosed by clinical, ultrasound or autopsy cases. NIHK cases have large etiological heterogeneity due to the combination of abnormalities in each system, which we will not discuss. For the etiology of IHK cases, only 1 article  reportedIn IHK cases, the subgroups with different amounts of amniotic fluid were subjected to sensitivity analysis, the mortality rate was 34% in oligohydramnios and 7% in the normal amniotic fluid group after sensitivity analysis, there was no deaths from IHK with polyhydramnios. Subgroup analysis showed that the mortality rate varied greatly with different amount of amniotic fluid and the results were statistically significant. Among NIHK cases, the mortality rate was 46% in the oligohydramnios group and 26% in the normal amniotic fluid group, and only one article reported the occurrence of fetal death in polyhydramnios, with a mortality rate of 13%. The large difference in mortality among the groups indicated that the amount of amniotic fluid was an important factor affecting fetal mortality.The mortality rate in IHK was 36% in fetuses with enlarged kidneys and 7% in those with normal kidney size. No deaths were reported in fetuses with small kidney development. In the cases of NIHK, due to the limited number of relevant literature, the sensitivity analysis had little effect on the results of subgroup analysis, and the results were relatively stable. Therefore, the mortality rate of kidney enlargement was 55%, that of normal kidney was 22%, and that of relatively developed kidney was 3%. The mortality of NIHK was higher than that of IHK.Termination (TOP) rates were 21% in IHK and 63% in NIHK. Although there was a large heterogeneity of TOP in both groups, the sensitivity analysis found that the results were relatively stable and meaningful, so the literature was not eliminated. In addition, the TOP probability is also affected by the results of prenatal diagnosis, which is partly related to the amniotic fluid volume and renal development changes suggested by ultrasound during pregnancy. Another part of the reason is that some pregnant women cannot accept further examination due to the limitation of previous diagnostic technology, family history of patients or some other subjective factors, and the attitude or legal requirements of hospitals in different countries on the behavior of pregnancy termination are not consistent, resulting in certain differences in the TOP rate in different regions. In addition, our assessment of the prognosis of HK is more focused on intrauterine death (IUD) and neonatal death (ND). In well-followed studies, the death of childhood kidney disease is also more significant for the long-term prognosis of IHK. No intrauterine deaths have been reported in IHK cases. Intrauterine mortality was 2% in NIHK cases. The neonatal mortality in IHK was 13%. NIHK has a large heterogeneity of neonatal mortality. After sensitivity analysis, 1study was excluded, in which 2 neonatal deaths were postoperative deaths of diaphragmatic hernia in the neonatal period. We were not sure whether the death was due to the disease itself or surgical complications, which led to unstable results, so it was no longer considered for inclusion, and the neonatal mortality was re-evaluated to be about 15%. 1study each reported childhood mortality in IHK and NIHK cases, with mortality rates of 10% and 3%, respectively. The childhood mortality rate of IHK is about three times higher than that of NIHK, which may not be the same as we expected. The main reason is that the number of literature is too small to conduct meta-analysis, and the number of childhood deaths reported by only one article is not enough to describe the real mortality rate. Secondly, we can also understand that NIHK patients with poor prognosis died before childhood due to their own combined abnormalities of other systems, both intrarenal and extrarenal. In addition, the follow-up of childhood deaths requires a long period of time, and different duration of follow-up also affects the long-term mortality results. Therefore, we may need more articles with long-term follow-up to understand the long-term survival and mortality of this disease. Not all survivors are healthy. 22% of IHK survivors are still likely to face hypertension, diabetes, renal failure, dialysis, kidney transplantation, etc., compared with 29% of NIHK survivors.Fetuses with poor or absent CMD have a higher mortality rate of 65%. Cortico-medullary differentiation, a decrease in medullary and cortical thickness, is a hallmark of renal dysplasia , as a reThis meta-analysis comprehensively covered the etiology of IHK, the incidence of HEK monogenic etiologies, mortality, and prognostic healthy survival, disease survival, and prognostic survival factors. The main limitation of the current study is that most of the studies included were retrospective designs and some of the outcomes evaluated were reported only in a limited proportion of the included studies. In addition, as not all cases in the included studies were genetically tested, and not all were routinely measured kidney length, CMD, were only tested or measured when abnormalities were apparent, there may be selection bias. Nonetheless, the available data are sufficient to draw the conclusions.HNF1B variant. Oligohydramnios, renal enlargement and CMD with two or more items increase the possibility of poor prognosis. We currently recommend a detailed ultrasound scan of the HEK fetus, measuring kidney length, CMD in addition to the routine measurement of amniotic fluid, and periodically rechecking for changes. Interventional prenatal diagnosis is performed, including chromosome karyotype and CMA or CNV-seq, and WES testing is feasible when necessary, especially if the karyotype and CMA results are negative, do not ignore the possibility of monogenic etiologies Combined with various indicators, comprehensive evaluation of fetal prognosis, avoid unnecessary termination of pregnancy, and also reduce the birth of defects to the greatest extent.The positive rate of karyotype was 20% in HEK and 22% in NIHK. The positive rate of CMA was 32% in NIHK and 59% in IHK. The positive rate of IHK monogenic etiologies was 31%. The most common etiology of IHK is the


The liver keeps haematological parameters normal and preserves haemostasis by storing iron, vitamin B-12, and, folic acid, necessary for healthy haematopoiesis. Anaemia of various aetiologies affects approximately 75% of chronic liver disease (CLD) patients, specifically caused by iron deficiency, hypersplenism, chronic diseases, autoimmune haemolysis, folic acid deficiency, aplasticity, and as a side effect of antiviral drugs. This study sought to observe the derangements in haematological parameters in patients with CLD, analyse the spectrum of anaemia in patients with CLD, and predict CLD outcomes utilizing Child-Pugh Score. This cross-sectional observational research was carried out in the Department of General Medicine, Himalayan Institute of Medical Sciences (HIMS), Dehradun, India over the course of a year. The patients with CLD who were admitted to the ward participated in the study. Most patient’s blood pictures reported normocytic normochromic with thrombocytopenia (TCP) (28.7%), macrocytic hypochromic with TCP (26%), microcytic hypochromic with TCP (13.3%) and macrocytic normochromic with TCP (9.3%). The incidence of anaemia was 85.3%: mild in 12.7% patients, moderate in 55.3% patients, and severe in 17.3% patients. Interestingly, this study also builds upon others suggesting that 85.9% of CLD patients have Class C Child-Pugh Score. The liver is the most expansive and complex organ in terms of its functioning. In the human body, it performs a wide variety of activities including but not limited to the breakdown of carbohydrates, proteins, and lipids, neutralisation of a wide variety of toxins, metabolization of exogenous drugs and hormones, production of plasma proteins, and maintenance of the immune system, specifically the Kpuffer cells. In foetal life, liver is the major site of haematopoiesis, contrary to postnatal life, where it maintains haematological haemostasis. It further plays a significant role in the regulation of blood pressure and cascades secreting clotting factors and inhibitors, in addition to serving as a storage depot for iron, folic acid, and vitamin B-12 [Chronic liver disease (CLD) causes slow wear and tear of the liver echotexture leading to fibrosis and cirrhosis, both being conferred end stages of the illness. Concurrent complications such as bleeding and infection can lead to an increase in morbidity and death rates. Sometimes, it is connected with haematological abnormalities. CLD can have a myriad of underlying causes including but not limited to dietary deficiencies, bleeding, excessive use of alcohol, and abnormalities in the production of hepatic proteins or proteins involved in blood formation or coagulation. Overall CLD is linked to a broad spectrum of different haematological disorders . More spAnaemia in CLD is caused by iron deficiency, hypersplenism, chronic disease anaemia, autoimmune haemolytic anaemia, folic acid deficiency, aplastic anaemia, and as a side effect of antiviral drugs . CLD comParticipantsThis cross‑sectional study was reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement, that is, the guidelines for reporting observational studies. It was carried out in the Department of General Medicine at the Himalayan Institute of Medical Sciences (HIMS), Swami Rama Himalayan University (SRHU) in Swami Ram Nagar, Dehradun, India. Ethics approval (Project no: SRHU/HIMS/RC/2023/110) was granted prior to recruitment, all participants provided written informed consent, and we adhered to our protocol to the Declaration of Helsinki. The patients with CLD, who were admitted to the HIMS in-patient ward participated in the observational study. Subjects were recruited for detailed history, thorough clinical examination, and investigations as per the working proforma. Patients more than 18 years age belonging to either gender with CLD due to: chronic hepatitis-C, chronic hepatitis-B, non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis, autoimmune hepatitis, Wilson’s disease, and haemochromatosis were included in the study. However, patients diagnosed with acute hepatitis, drug-induced acute hepatic injury, fulminant hepatic failure, malignancy at the time of presentation, and jaundice associated with seasonal viral infection were excluded from the study sample.Study design9/L. The outcome of the patients was categorised as favourable (improved and discharged) or unfavourable . The outcome groups so formed were compared with regard to haematological and biochemical parameters.Structured data collection form was used to record patient data as follows: Haematological parameters: haemoglobin, total leucocyte count (TLC), differential leucocyte count (DLC), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), reticulocyte count, platelet count and mean platelet volume, Red cell distribution width (RDW). All cases of a CLD diagnosed in the Medicine out-patient door (OPD) and inpatient door (IPD) satisfying the inclusion criteria were taken. CLD was defined if the following parameters were fulfilled . Anaemia is categorized as (1) mild anaemia ranges <13 g/dL to 10 g/dL, (2) moderate between 7 g/dL to 10 g/dL, and (3) severe anaemia below 7 g/dL. Patients were diagnosed with TCP if their platelet count was less than 150 x 10Statistical analysisExcel for Microsoft Windows was used to enter the data, and the Statistical Package for Social Sciences (SPSS) was to do the statistical analysis. To investigate the distribution of a number of categorical and quantitative variables, a descriptive statistical analysis was carried out in the SPSS. To summarise categorical data , we used frequency (%) and mean ± standard deviation. One-way Analysis of variance (ANOVA) was used to evaluate whether there is a statistically significant difference between categories of anaemia . If the p-value is less than 0.05, the experiment is regarded to be statistically significant.Participants demographicsMost patients belonged to the age group of 41-50 years (30%) and 51-60 years (28%). This was followed by 31-40 years (18%), 61-70 years (12%), 22-30 years (8.7%) and 71-80 years (3.3%). The mean age was 48.23 ± 12.15 years. Males and females were 88% and 12% respectively. From an occupation viewpoint, the majority of the patients were shopkeepers (27.3%), retired personnel (16%), and farmers (15.3%).Haematological profileTable Table Table Figure This study has three primary objectives: to observe the derangements in haematological and biochemical parameters in patients with CLD, to analyse the spectrum of anaemia in patients with CLD, and to establish the association between the aetiology of CLD, and the type and severity of anaemia. Our results showed a haematological profile of CLD patients largely depends upon the aetiology and that has exhibited a mixed pattern in most cases. Most blood parameters, including the red blood cells, white blood cells, or haemostasis parameters although deter, only Hb (g/dL) significantly plummets across anaemia severity. And Child-Pugh score suggests advanced hepatic dysfunction across most CLD cases.Pathophysiology: haematological derangement in CLDPatients who suffer from alcoholic liver cirrhosis frequently present with abnormalities in their haematological parameters. There are multiple causes that contribute to the development of aberrant haematological indices. These variables include portal hypertension-induced sequestration, changes in bone marrow stimulating factors, and virus and toxin-induced bone marrow suppression. Consuming excessive amounts of alcohol produces direct suppression of the bone marrow, which in turn leads to harmful effects on the blood cell lines. Indirectly, it interferes with the patient's nutritional biology, which ultimately leads to the generation of cells that are not fully mature in their functions. Alcohol consumption creates complex aberrations in people with Haematological indices, which can lead to lethal consequences and an increased risk of death for these patients . AbnormaComparison to literature: haematological derangements in CLDIn our study, the majority of the patient’s blood picture showed normocytic normochromic with TCP (28.7%), macrocytic hypochromic with TCP (26%), microcytic hypochromic with TCP 13.3%) and macrocytic normochromic with TCP 9.3%). In our study, the majority of patients have the normocytic normochromic type of anaemia which is the most common anemia found in alcoholics followed by macrocytic hypochromic anaemia. It was found that TCP is almost always present in all patients of CLD, irrespective of the etiology. Similar results were seen in a study conducted by Tara et al. .3%. In o, 42.6% o.3% and m3, while the counts of 89% of patients were normal . Leucocytosis was found to be present in 3.3% of the individuals who were examined. There was no link found between the TLCs and either the presence of splenomegaly or the amount of time it took for the patient to bleed. Neither of these factors affected the length of time it took for the patient to bleed. Because leukopenia and leucocytosis are more prevalent in persons who have infections, such as urinary tract infections and spontaneous bacterial peritonitis, it is essential to understand the reasons behind why this is the case. 20.9% of patients displayed leucocytosis (TLC>11 x 109), whereas 17.4% of patients demonstrated leukopenia, as stated by Khan et al. [9). According to Kaur et al. [3) and the mean platelet count (x103/micro-L) were, respectively, 11,445 and 151. Both of these numbers are expressed in millimetres per cubic inch. Out of a total of 90 individuals, leucocytosis was seen in 41 patients (45.6%), whilst leukopenia was identified in 14 patients (15.6%). In our study, an association of MCV with the severity of anaemia was found to be statistically not significant. In the study done by Kaur et al. [In our study, an association of TLC with the severity of anaemia was found to be statistically not significant. Patients in the normal range have TLC (9.36 ± 2.98), mild anaemia with TLC (8.47 ± 2.98), moderate anaemia with TLC (10.10 ± 3.87) and severe anaemia with TLC (9.59 ± 3.96). According to the findings of research that was carried out by Sambyal et al. , the totr et al. , the mear et al. , microcy3 according to Jain et al. [In our study, an association of platelet count with the severity of anaemia was found to be statistically not significant. We found that the patient having normocytic anaemia was also found to have more TCP. So, the percentage and the severity of TCP are present in the normocytic type of anaemia in patients of CLD, irrespective of the ethology. Maximum patients of CLD were found to have TCP, irrespective of ethology and no association was found with the severity of anaemia. In our study, 60 % of the patients have TCP and in a similar study conducted by Kaur et al.  TCP was n et al. .As a whole, it is important to underscore that the results of this study should not be interpreted as to demonstrate a clear pattern of specific haematological derangements. Indeed, there is still a need to evaluate the haematological profile, specifically in the context of alcohol-related liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD); however, it will take time until that happens.Clinical implications and managementWhilst evaluating CLD, non-invasive assessment of fibrosis and cirrhosis is highly demanded. Further, identification of haematological derangements in NAFLD, conditions caused by a build-up of fat in the liver, specifically NASH is needed. Additionally, the search for novel medications to improve the prognosis of NAFLD is urgently warranted.Limitations and future directionsThe cause of anaemia in individual patients has not been extensively studied which could make a difference in the treatment approach. This specifically pertains to iron, vitamin B12, and folic acid deficiencies. A more type-specific analysis would underpin the treatment plan per se. Also, multivariate analysis of the co-variates could add a new perspective to anaemia in CLD. Since data collection was explicitly based on hospital questionnaires, the paucity of some key parameters constrained us from performing further multivariate analysis. Under-representation of women (only 12%) in our sample can be explained by the fact that CLD prevalence is much larger in men than women, perhaps due to higher alcohol consumption. Further studies with a greater number of patients, particularly of different ethnicities from across the globe will surely bolster the results of this study.The complete blood count had shown that the majority of patients had anaemia, leukopenia, and TCP. The incidence of anaemia was 85.3%: mild in 12.7% patients, moderate in 55.3% patients, and severe in 17.3% patients. Interestingly, this study also builds upon others suggesting that 85.9% of CLD patients have Class C Child-Pugh Score.


Antibiotics are double-edged swords. Although antibiotics are used to inhibit pathogenic bacteria, they also run the risk of destroying some of the healthy bacteria in our bodies. We examined the effect of penicillin on the organism through a microarray dataset, after which 12 genes related to immuno-inflammatory pathways were selected by reading the literature and validated using neomycin and ampicillin. The expression of genes was measured using qRT-PCR. Several genes were significantly overexpressed in antibiotic-treated mice, including CD74 and SAA2 in intestinal tissues that remained extremely expressed after natural recovery. Moreover, transplantation of fecal microbiota from healthy mice to antibiotic-treated mice was made, where GZMB, CD3G, H2-AA, PSMB9, CD74, and SAA1 were greatly expressed; however, SAA2 was downregulated and normal expression was restored, and in liver tissue, SAA1, SAA2, SAA3 were extremely expressed. After the addition of vitamin C, which has positive effects in several aspects, to the fecal microbiota transplantation, in the intestinal tissues, the genes that were highly expressed after the fecal microbiota transplantation effectively reduced their expression, and the unaffected genes remained normally expressed, but the CD74 gene remained highly expressed. In liver tissues, normally expressed genes were not affected, but the expression of SAA1 was reduced and the expression of SAA3 was increased. In other words, fecal microbiota transplantation did not necessarily bring about a positive effect of gene expression restoration, but the addition of vitamin C effectively reduced the effects of fecal microbiota transplantation and regulated the balance of the immune system. Antibiotics (ABX) are double-edged swords. Although antibiotics are used to inhibit pathogenic bacteria, they also run the risk of destroying some of the healthy bacteria in our bodies. The intestinal microbiota attaches to and colonizes the gut and participates in intestinal food processing and host nutrition. The intestinal microbiota also promote the maturation of the intestinal epithelium and maintains the integrity of the body’s immune system and natural barrier function . SimilarAfter antibiotic treatment, major defects in the production and activation of innate immune cells are demonstrated. The dysbiotic microbiota then translocates to colonization sites in the gut, which can lead to chronic periepithelial inflammation with barrier leakage, triggering a local inflammatory response or systemic immunity .via a different route of administration, thereby restoring the microbial ecosystem in the gut  is a gut therapy in which bacteria from the feces of a healthy donor are transferred to the patient  the gut . FMT has the gut . FMT is  the gut .Helicobacter pylori infection and gastric cancer by reactivating vitamin E and glutathione through its cytoprotective antioxidant effect in the stomach and by inhibiting endogenous N-nitrosylation and reducing the toxic effects of ingested nitrosodimethylamine and heterocyclic amines  database is a gene expression database created and maintained by the National Center for Biotechnology Information NCBI. We selected the data set with the registration number analysis .Fifteen 4-week-old C57BL/6J male mice (15∼25g) were purchased from SPF(Beijing) BIOTECHNOLOGY Co., Ltd. and housed individually in an experimental environment at 23 °C with a 12-hour light/dark cycle. During the experiment, mice had free access to sterilized food and water. After 10 days of acclimation, three mice were used as a control group (NO-ABX) for group N. Twelve mice received antibiotics for 21 days and were later randomly divided into four groups of three mice each in a cage for groups ABX, F, VF, and NS. The ABX group was executed by cervical dislocation after receiving antibiotics for 21 days, and intestinal and liver tissue samples were frozen in liquid nitrogen and stored at −80 °C. The NS group received antibiotics for 21 days followed by saline for 21 days, while the F group received fecal microbial transplantation for 21 days and the VF group received fecal microbial transplantation and vitamin C for 21 days. On day 42 of the experiment, mice in the N, F, VF, and NS groups were executed by cervical dislocation, and intestinal and liver tissue samples were frozen in liquid nitrogen and stored at −80 °C. The collected tissue samples were subjected to quantitative real-time PCR . All aniThe Research Ethics Committee of Chongqing University of Posts and Telecommunications approved all experimental procedures .ad libitum by the mice, and changed once a day for 21 days of administration.Single antibiotics have limited clearance efficiency for complex microbial ecosystems . ChristoThe role of FMT is also limited in that it allows the establishment of only a small fraction of the human bacterial community . Korry JTotal RNA was extracted from collected intestinal and liver tissues using TRIzol reagent  according to the manufacturer’s protocol, and the purity of RNA was measured using spectrophotometric analysis . Next, RNA was reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis Kit . The quantitative real-time PCR (qRT-PCR) analysis was performed using SYBRPRIME qPCR Kit  and Bio-Rad iQ5 software . GAPDH was analyzed in each sample to normalize expression. Three biological replicates were performed for each gene, and each replicate contained three technical replicates. The primers used in this study are listed in t-test, and differences between the three groups were analyzed using analysis of variance (ANOVA). GEO data were transformed from ENTRZ ID annotation to SYMBOL ID by R language, and after voom normalization by limma  (6.7 mg/L) from birth and three mice were born normally as control, ileum was collected at eight weeks of age, RNA was extracted, and the differences were analyzed, with —FC—>1 representing significant and P value < 0.05 representing statistically significant screening for differential genes. As shown in the volcano plot in vs. 12 down-regulated genes, and was plotted by hierarchical clustering , is recognized by CD4+ T cells (helper T cells), is expressed only on the surface of antigen-presenting cells such as macrophages, dendritic cells, and B cells, and is known to be involved primarily in the immune response. Low-dose penicillin alters the expression of immune-related genes in mice and disrupts the original balance of immune gene expression in the mouse ileum. A KEGG pathway enrichment analysis was then performed, as shown in To investigate the effect of antibiotics on intestinal gene expression, the ring see . Each coThree other normally grown mice and three mice receiving low penicillin injections had their livers collected at 30 weeks of age and RNA extracted. differential analysis revealed three upregulated genes, Neat1, Cirbp, and Rian, and five downregulated genes, Hspa1b, Ugt2b38, Hspb1, Cidec, and Slc10a2 . Enrichmin vitro migration and recruitment of a variety of cells, including T cells, and plays a role in a variety of biological processes including cancer and atherosclerosis. ccl5 also interacts with a variety of other receptors to regulate cellular activity, receptor and chemokine levels and maintain homeostasis in vivo  were highly expressed in the FMT group, with only SAA2 significantly lowly expressed, CD74 still highly expressed, and UBD largely unexpressed. For liver tissues, the expression of H2-DMA remained low, the expression of SAA1, SAA2, SAA3, and PSMB8 was significantly higher, H2-AA was significantly lower, and only GZMB, CD3G, and PSMB9 changed in a good direction  has been shown to have health benefits and to be involved in biological processes that support the immune system . VitaminTo investigate whether vitamin C could affect fecal microbiota transplantation, we designed an experimental group in which mice were transplanted with fecal microbes for 21 consecutive days after 21 days of Abx treatment and given the same dose of vitamin C. The qPCR results showed that for intestinal tissues, GZMB, CD3G, H2-AA, PSMB9, SAA1 had better results and were closer to the genes in the normal group, reducing the expression of their highly expressed genes in the F group, these genes function in Granzyme Pathway, Intrinsic Pathway for Apoptosis, Programmed Cell Death, Immune response NFAT in immune response, TCR Signaling (Qiagen), Class I MHC mediated antigen processing and presentation, Cytokine Signaling in Immune system, among other pathways. The addition of vitamin C had no significant effect on CD74, UBD, PSMB8, SAA3, H2-DMA, SAA2, and CCL5, but it did not cause worse effects either. In liver tissues, more favorable effects were found for GZMB, CD3G, H2-AA, PSMB8, SAA1, and less favorable effects for the other seven genes. It is also important to note that the expression of genes in both tissues was higher in the intestine than in the liver for group F genes, and the same was true after the addition of vitamin C. This suggests that vitamin C acts directly in the intestine and more weakly in the liver. In conclusion, for intestinal tissues, the introduction of vitamin C restored the genes more to normal levels, and for liver tissues, vitamin C mostly reduced the expression of highly expressed genes .Intestinal microbes are microorganisms that reside in the intestinal and play an important role in the development of metabolic diseases, cancer, and neurological disorders, and a large number of factors have an impact on the number and diversity of intestinal microbes, such as lifestyle, dietary intake, and medications . Recent versus liver differentially expressed genes under the influence of low-dose penicillin in the GSE58087 microarray dataset. The differential genes were found to be mostly enriched in immune responses such as antibody treatment and expression, interferon IFN-γ response, and exogenous peptide antibody treatment and expression in intestinal tissues, suggesting that antibiotics mediate the occurrence of intestinal immune responses through intestinal microbes. However, the nine differential genes identified in liver tissue did not overlap with those in ileal tissue: Neat1, Cirbp, Rian/Meg8, Hspa1b, Ugt2b38, Hspb1, Cidec, and Slc10a2, and were mainly enriched in protein folding. This may be due to the direct effect of antibiotics on the intestine and their impact on the intestinal flora, which in turn indirectly affects the liver through the ‘enterohepatic axis’. We mainly investigated the changes in immune genes after antibiotic-induced inflammation and the subsequent recovery, so we selected 12 key genes for subsequent validation analysis after reviewing the literature and related literature.In this study, we investigated the effect of antibiotics on the differential expression of relevant immune-inflammatory genes in the intestine and liver. First, we screened the ileal β9 subunit plays an important role in the human leukocyte antigen class I (HLAI) delivery system (In intestinal tissues, all 10 genes were significantly overexpressed, except H2-AA with H2-DMA. After natural recovery by normal saline gavage, H2-AA remained lowly expressed, CD74 and SAA2 remained highly expressed but largely decreased gradually, while the other genes largely returned to normal levels. It is suggested that antibiotics activate various inflammatory signals by affecting intestinal microbes. CD74 differs most from SAA2. CD74 is an immune response-related transcription factor involved in the inflammatory response and hematopoietic development and function, and activation of CD74 causes transcriptional activation, while the genes it regulates include genes of the NF-κB pathway and cell death, genes related to immune cell motility and leukocyte recruitment, and genes for blood cell interactions and adhesion are all rapidly upregulated when it is activated . Circulay system , suggestTo further confirm the effect of intestinal microbes on gene expression, we attempted to restore the microbiota of mice by fecal microbiota transplantation after ABX treatment of mice. A study showed that in mice treated with FMT after antibiotics and chemotherapy, dysregulation of the intestinal microbiota was rapidly corrected and no significant pathogenic bacteria appeared in the intestine. Thus, FMT restores a healthy and diverse intestinal microbiota that may potentially prevent the emergence of pathogenic bacteria and bacterial translocation from the gastrointestinal tract to the bloodstream . HoweverVitamin C plays an important role in several ways; it is a potent major non-enzymatic antioxidant in plasma that reacts with and scavenges free radicals, thereby inhibiting chain reactions and thus preventing lipid peroxidation damage , VitaminIn this study we determined that antibiotics mediated gene expression in the intestinal and liver by modulating intestinal microbes; therefore, incorporating the intestinal and liver response is needed when using antibiotics in the future. We also found that fecal microbial transplantation did not necessarily have a positive effect on intestinal microbiota recovery, but the addition of vitamin C did have a positive effect, but more factors still need to be explored. Again this study has some limitations, the selection number of genes is too small and does not reflect the complete changes in the intestinal and liver. There was no sampling at multiple time points, and it is not particularly clear how deeply the intestinal microbiota affects the genes. Due to experimental errors, two extreme data were removed when conducting the mice experiments, resulting in only three mice per group and reduced reliability of the experiment. In future studies, we hope to improve the process and content.10.7717/peerj.15356/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15356/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15356/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15356/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.15356/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.15356/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.15356/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.15356/supp-8Supplemental Information 8Click here for additional data file.