The Effects of Composite Alkali-Stored Spent Hypsizygus marmoreus Substrate on Carcass Quality, Rumen Fermentation, and Rumen Microbial Diversity in Goats.

The objective of this study was to investigate the effects of composite alkali-stored spent Hypsizygus marmoreus substrate (SHMS) on carcass quality, rumen fermentation, and rumen microbial diversity in goats. Twenty-four 6-month-old Chuanzhong black goats with similar body weights (20 ± 5 kg) were selected and randomly divided into four groups (n = 6 per group) and received four treatments: 0% (control group, CG); 20% (low-addition group, LG); 30% (moderate-addition group, MG); and 40% (high-addition group, HG) of SHMS-replaced silage corn and oat hay. The experiment lasted for 74 days (including a 14 d adaptation period and a 60 d treatment period). The results of this study showed that MG and HG significantly improved the marble score of goat meat (p < 0.05). The flesh color score significantly increased in each group (p < 0.05). The fat color scores significantly increased in LG and MG (p < 0.05). There were no significant effects on the pH value or shear force of the longissimus dorsi in each group (p > 0.05). The cooking loss in MG was higher than that in CG (p < 0.05). The histidine and tyrosine contents in each group of muscles significantly increased (p < 0.05), with no significant effect on fatty acids (p > 0.05). The rumen pH of MG significantly decreased (p < 0.05), while the total volatile fatty acids (TVFAs) and ammoniacal nitrogen (NH3-N) increased by 44.63% and 54.50%, respectively. The addition of the SHMS altered both the alpha and beta diversities of the rumen microbiota and significant differences in the composition and structure of the four microbial communities. The dominant bacterial phylum in each group were Firmicutes and Bacteroidetes, with Prevotella 1 as the dominant bacterial genus. Correlation analysis revealed that rumen bacteria are closely related to the animal carcass quality and rumen fermentation. In the PICRUSt prediction, 21 significantly different pathways were found, and the correlation network showed a positive correlation between the Prevotella 1 and 7 metabolic pathways, while the C5-branched dibasic acid metabolism was positively correlated with nine bacteria. In summary, feeding goats with an SHMS diet can improve the carcass quality, promote rumen fermentation, and alter the microbial structure. The research results can provide a scientific reference for the utilization of SHMS as feed in the goat industry.

Analysis of Rumen Degradation Characteristics, Attached Microbial Community, and Cellulase Activity Changes of Garlic Skin and Artemisia argyi Stalk.

The purpose of this study was to study the chemical composition, rumen degradation characteristics, surface attached microbial community and cellulase activity of garlic skin (GS) and Artemisia argyi stalk (AS), in order to explain their feeding value. Four 14-month-old healthy Min Dong male goats with permanent rumen fistula were selected as experimental animals. The rumen degradation characteristics of GS and AS were determined by using the nylon bag method, and the bacterial composition, cellulase activity and their relationship on the surface of the two groups were analyzed with high-throughput sequencing of 16S rRNA gene. The results showed that in GS and AS, the effective degradation rate (ED) values of dry matter (DM) were 42.53% and 37.12%, the ED values of crude protein (CP) were 37.19% and 43.38%, the ED values of neutral detergent fiber (NDF) were 36.83% and 36.23%, and the ED values of acid detergent fiber (ADF) were 33.81% and 34.77%. During rumen degradation, the richness and evenness of bacteria attached to the AS surface were higher. At the phylum level, Bacteroidetes and Firmicutes were always the main rumen bacteria in the two groups. At the genus level, fiber-degrading bacteria such as Prevotella, Treponema, and Ruminococcus showed higher levels in GS (p < 0.05). Compared with GS, the activity of ß-glucosidase (BG enzyme), endo-ß-1,4-glucanase (C1 enzyme), exo-ß-1,4-glucanase (Cx enzyme) and neutral xylanase (NEX enzyme) attached to AS surface showed a higher trend. Correlation analysis showed that the relative abundance of Succinivibrio and Rikenellaceae_RC9_gut_group was positively correlated with the rumen degradability of nutrients in GS, and the relative abundance of Christensenellaceae R-7_group, Succinivibrio and Ruminococcus was positively correlated with the rumen degradability of nutrients in AS. The conclusion of this study shows that AS has more potential to become ruminant roughage than GS. In addition, this study also revealed the relationship between cellulase activity and bacteria, which provided new information for us to better analyze the effects of GS and AS on the rumen of ruminants and provided an important theoretical basis for the development and utilization of agricultural by-products.

The Effects of Temperature and Humidity Index on Growth Performance, Colon Microbiota, and Serum Metabolome of Ira Rabbits.

This study investigates the effects of different THI values on growth performance, intestinal microbes, and serum metabolism in meat rabbits. The results showed that there were significant differences in THI in different location regions of the rabbit house. The high-THI group (HG) could significantly reduce average daily gain and average daily feed intake in Ira rabbits (p < 0.05). The low-THI group (LG) significantly increased the relative abundance of Blautia (p < 0.05). The HG significantly increased the relative abundance of Lachnospiraceae NK4A136 group and reduced bacterial community interaction (p < 0.05). The cytokine-cytokine receptor interactions, nuclear factor kappa B signaling pathway, and toll-like receptor signaling pathway in each rabbit's gut were activated when the THI was 26.14 (p < 0.05). Metabolic pathways such as the phenylalanine, tyrosine, and tryptophan biosynthesis and phenylalanine metabolisms were activated when the THI was 27.25 (p < 0.05). Meanwhile, the TRPV3 and NGF genes that were associated with heat sensitivity were significantly upregulated (p < 0.05). In addition, five metabolites were found to be able to predict THI levels in the environment with an accuracy of 91.7%. In summary, a THI of 26.14 is more suitable for the growth of meat rabbits than a THI of 27.25, providing a reference for the efficient feeding of meat rabbits.

House ammonia exposure causes alterations in microbiota, transcriptome, and metabolome of rabbits.

Introduction: Pollutant gas emissions in the current production system of the livestock industry have negative influences on environment as well as the health of farm staffs and animals. Although ammonia (NH3) is considered as the primary and harmful gas pollutant in the rabbit farm, less investigation has performed to determine the toxic effects of house ammonia exposure on rabbit in the commercial confined barn. Methods: In this study, we performed multi-omics analysis on rabbits exposed to high and low concentration of house ammonia under similar environmental conditions to unravel the alterations in nasal and colonic microbiota, pulmonary and colonic gene expression, and muscular metabolic profile. Results and discussion: The results showed that house ammonia exposure notably affected microbial structure, composition, and functional capacity in both nasal and colon, which may impact on local immune responses and inflammatory processes. Transcriptome analysis indicated that genes related to cell death (MCL1, TMBIM6, HSPB1, and CD74) and immune response (CDC42, LAMTOR5, VAMP8, and CTSB) were differentially expressed in the lung, and colonic genes associated with redox state (CAT, SELENBP1, GLUD1, and ALDH1A1) were significantly up-regulated. Several key differentially abundant metabolites such as L-glutamic acid, L-glutamine, L-ornithine, oxoglutaric acid, and isocitric acid were identified in muscle metabolome, which could denote house ammonia exposure perturbed amino acids, nucleotides, and energy metabolism. In addition, the widespread and strong inter-system interplay were uncovered in the integrative correlation network, and central features were confirmed by in vitro experiments. Our findings disclose the comprehensive evidence for the deleterious effects of house ammonia exposure on rabbit and provide valuable information for understanding the underlying impairment mechanisms.

[Holocarboxylase synthetase deficiency induced by HLCS gene mutations: a rare disease study]. / ç½•è§ç— ç ”ç©¶ï¼šHLCSåŸºå› çªå˜è‡´å ¨ç¾§åŒ–é ¶åˆæˆé ¶ç¼ºä¹ç—‡.

A boy, aged 16 months, attended the hospital due to head and facial erythema for 15 months and vulva erythema for 10 months with aggravation for 5 days. The boy developed perioral and periocular erythema in the neonatal period and had erythema and papules with desquamation and erosion in the neck, armpit, and trigone of vulva in infancy. Blood gas analysis showed metabolic acidosis; the analysis of amino acid and acylcarnitine profiles for inherited metabolic diseases and the analysis of organic acid in urine suggested multiple carboxylase deficiency; genetic testing showed a homozygous mutation of c.1522C>T(p.R508W) in the HLCS gene. Finally the boy was diagnosed with holocarboxylase synthetase deficiency and achieved a good clinical outcome after oral biotin treatment. This article analyzes the clinical data of a child with holocarboxylase synthetase deficiency and summarizes the etiology, diagnosis, and treatment of this child, so as to provide ideas for clinicians to diagnose this rare disease.

Lauromacrogol foam injection under ultrasonic guidance for pediatric lip venous malformations.

OBJECTIVE: To evaluate the efficacy and safety of lauromacrogol foam sclerotherapy in the treatment of children with lip venous malformation. METHODS: Fifty-two children (27 males and 25 females) aged from 6 months to 17 years with lip VM who underwent lauromacrogol foam injection with ultrasonic guidance from July 2018 to December 2020 in our hospital were retrospectively recruited for this study. All the children were examined by MRI, ultrasound, blood routine and coagulation before operation. We were guided by ultrasound to locate the blood flow area (nests), injecting lauromacrogol foam to fill the venous malformation. The follow-up time was 14.31 ± 5.96 (6-24) months. Follow-up items include clinical manifestations, imaging data, efficacy and complications. RESULTS: This group of children was treated 3-5 times, an average of 4 times/case. The total effective rate was 90.38%. Pain in 4 cases, fever in 4 cases, infection in 2 cases, ulcer in 1 case. There were no serious complications such as cardiopulmonary accident. CONCLUSION: Ultrasound guiding foam sclerotherapy with lauromacrogol is effective and safe for children with lip venous malformation.

Status of linusorbs in cold-pressed flaxseed oil during oxidation and their response toward antioxidants.

Linusorbs (LOs, cyclolinopeptides) are a class of naturally occurring cyclic hydrophobic peptides found in flaxseed oil, whose oxidation states indicate the oxidative stability and bitterness of flaxseed oil. Subjected to 63 °C accelerated oxidation, most Met-containing LOs in cold-pressed flaxseed oil entirely depleted by the 6th day except CLP, and MetO2-containing LOs became the dominant ones. However, no MetO2 form of Trp-containing LOs, such as CLD, CLF and CLG, were detected. Given their oxidative kinetics, methionine sulfoxide (MetO) residue in some LOs was less sensitive toward oxidation in the presence of Trp (Tryptophan) group, and the oxidative stability of Met-containing LOs was CLP < CLB < CLL ≈ CLM < CLO, as compared to MetO-containing LOs: CLD < CLE < CLC < CLF ≈ CLG. When antioxidant was added into cold-pressed flaxseed oil to assess the additives' antioxidant effect, no significant difference was observed on oil oxidative indices in early stage except α-tocopherol, where they vary dramatically in suppressing Met oxidation of LOs: L-AP (L-ascorbyl palmitate) > TBHQ (tert-butyl hydroquinone) > Î³-tocopherol > carnosic acid > α-tocopherol. Besides its ability to suppress oxidation of Trp-containing LOs, L-AP also exhibits superior antioxidant effect on non-Trp-containing LOs due to its amphiphilic property. Due to the prooxidative effects of both α- and γ-tocopherol on LOs that contain Trp, it has been suggested that tocopherols may repair Trp residue on LOs, leading to increased tendency of MetO residues to oxidize. The findings of this research are critical for elucidating the antioxidative mechanism of LOs, which can further lead to the establishment of strategies in suppressing bitter after taste to produce high-quality flaxseed oil.

[Recurrent systemic sporadic rash for 10 years in a girl aged 11 years]. / 11å²å¥³å­©åå¤å ¨èº«æ•£åœ¨çš®ç–¹10å¹´.

A girl, aged 11 years, was admitted due to recurrent rash on the whole body and mucosa for 10 years, and typical rash was erythema at the perioral region, hand-foot joints, vulva, and perianal region, with blisters, erosions, and ulcers on the erythema. The girl was improved after zinc supplementation. Her younger brother had similar rash and medical history. The histopathological examination showed epidermal parakeratosis with mild hyperkeratosis, severe spongiform edema of the stratum corneum, significant proliferation of acanthocytes, and vacuolation of keratinocytes. The genetic testing revealed that both the girl and her younger brother had a homozygous mutation of c.1456(exon9)delG in the SLC39A4 gene, and thus the girl was diagnosed with acrodermatitis enteropathica. It is concluded that for children with recurrent rash on the limbs and at the perioral region, genetic testing should be performed as early as possible to make a confirmed diagnosis, and a sufficient dose of zinc supplementation should be given, while the levels of trace elements such as blood zinc should be regularly monitored.

Safety behaviors and job satisfaction during the pandemic: The mediating roles of uncertainty and managerial commitment.

INTRODUCTION: As the Covid-19 pandemic affects the world, disruptions to work routines impose a psychological burden on people, and thus can affect their job performance and well-being. We conducted an empirical study to explore the links between the experience of Covid-19 and workers' safety behaviors and well-being outcome of job satisfaction. METHOD: Structural equation modelling (SEM) with a sample of 515 safety workers was conducted to simultaneously test the links among these constructs. RESULTS: Experience of Covid-19 was associated with lower employee job satisfaction, explained by higher psychological uncertainty and decreased perception of managerial commitment to safety. Notably, contrasting pathways from experience of Covid-19 to safety behaviors were found. On the one hand, higher psychological uncertainty caused by the pandemic was associated with lower perceptions of managerial safety commitment; and lower perceived managerial safety commitment was linked to reduced safety compliance and safety participation. On the other hand, experience of Covid-19 in this study showed unexpected positive direct links with safety behaviors, which might be explained by workers' enhanced safety knowledge, motivation, and status of mindfulness due to Covid-19 related safety instructions and communications. CONCLUSIONS: This exploratory study helps to deepen the understanding of workplace safety and well-being in the context of pandemic and in times of uncertainty. PRACTICAL APPLICATIONS: The practical insights are useful for applying appropriate strategies for managing the Covid-19 crisis, coping with uncertainties, and building a healthier and safer workplace in the long run.

A Stock Selection Model of Image Classification Method Based on Convolutional Neural Network.

With the development of artificial intelligence technology, an increasing number of researchers try to apply different machine learning and deep learning methods to quantitative trading fields to obtain more stable and efficient trading models. As a typical quantitative trading strategy, stock selection has also attracted a lot of attention. There are many studies and applications on stock selection. However, the existing research and application cannot meet the continuous expansion of the scale and dimension of stock selection data set and cannot meet the needs in terms of efficiency and accuracy of stock selection. A convolutional neural network has been applied to image classification and achieved better results than the traditional methods. In this study, we first constructed a multifactor stock selection data set based on China's stock market. Then, we apply the convolutional neural network model to analyze stock selection data and select stocks. The main contribution of this study is that we build a stock multifactor data set, construct a "factor picture," and classify them by convolutional neural network to select stocks. This study also makes comparative experiments on the decision tree, support vector machine, and feedforward neural network in stock selection on the same data set constructed in this study. The results show that the stock selection method based on the convolutional neural network outperforms other methods in terms of the annual return, sharp ratio, and max drawdown.

Evidence of Guest-Guest Interaction in Clathrates Based on In Situ Raman Spectroscopy and Density Functional Theory.

Isotopes are ideal substances for studying the intermolecular interactions in clathrates by replacing the atoms without destroying the geometry structure. When methane (CH4) in the spatially homogeneous methane hydrate was replaced with deuterated methane (CD4), it showed a previously unrecognized strong anharmonic effect, identified by the Raman peak located at 1952.78 cm-1. This was assigned to a coupled overtone of C-D in 512 and 51262 cages on the basis of density functional theory. This coupling vibration was confirmed to be present also in methane hydrate by a peak around 3053.62 cm-1; its intensity is only 21.9% of that in the CD4 system. This coupled vibration may have been observed in previous studies, yet without any solid evidence of its detailed assignment. Our work could provide a tool for characterizing the intermolecular behavior in the guest-host system; the proposed method should also be employed universally for similar isotopic supramolecular compounds.

768-ary Laguerre-Gaussian-mode shift keying free-space optical communication based on convolutional neural networks.

Beyond orbital angular momentum of Laguerre-Gaussian (LG) modes, the radial index can also be exploited as information channel in free-space optical (FSO) communication to extend the communication capacity, resulting in the LG- shift keying (LG-SK) FSO communications. However, the recognition of radial index is critical and tough when the superposed high-order LG modes are disturbed by the atmospheric turbulences (ATs). In this paper, the convolutional neural network (CNN) is utilized to recognize both the azimuthal and radial index of superposed LG modes. We experimentally demonstrate the application of CNN model in a 10-meter 768-ary LG-SK FSO communication system at the AT of Cn2 = 1e-14 m-2/3. Based on the high recognition accuracy of the CNN model (>95%) in the scheme, a colorful image can be transmitted and the peak signal-to-noise ratio of the received image can exceed 35 dB. We anticipate that our results can stimulate further researches on the utilization of the potential applications of LG modes with non-zero radial index based on the artificial-intelligence-enhanced optoelectronic systems.

Phase Diagrams for sII Clathrate Hydrates of CO2 from First-Principles Thermodynamics.

Clathrate hydrates are crystalline solid compounds consisting of a water caged framework and guest molecules such as CH4, C2H6, and CO2. Understanding the phase equilibrium conditions of hydrates is significantly important for the industrial exploitation and experimental synthesis of hydrates. Based on the correct description of the intermolecular noncovalent interactions of clathrate hydrates with vdW-DF2, we studied the crystal structures and the chemical potential phase diagrams of sII hydrates encapsulated with CO2 molecules to provide a deep understanding of the stability mechanism of hydrates. Under the given p-T conditions, the partially occupied hydrates (136H2O·1CO2 and 136H2O·16CO2) and fully occupied hydrates (136H2O·24CO2) are thermodynamically stable, and the equilibrium temperature decreases as the relative CO2 chemical potential increases at the same pressure. We expect that the present study may provide vital information on the stability conditions of CO2 hydrates and trigger new experiments to establish an effective replacement strategy for CO2/CH4.

Varying degrees of homostructurality in a series of cocrystals of antimalarial drug 11-azaartemisinin with salicylic acids.

The X-ray structures of three new 1:1 pharmaceutical cocrystals of 11-azaartemisinin (11-Aza; systematic name: 1,5,9-trimethyl-14,15,16-trioxa-11-azatetracyclo[10.3.1.04,13.08,13]hexadecan-10-one, C15H23NO4) with bromo-substituted salicylic acids [namely, 5-bromo- (5-BrSalA, C7H5BrO3), 4-bromo- (4-BrSalA, C7H5BrO3) and 3,5-dibromosalicylic acid (3,5-Br2SalA, C7H4Br2O3)] are reported. All the structures are related to the parent 11-Aza:SalA cocrystal (monoclinic P21) reported previously. The 5-BrSalA analogue is isostructural with the parent, with lattice expansion along the c axis. The 4-BrSalA and 3,5-Br2SalA cocrystals retain the highly preserved 21 stacks of the molecular pairs, but these pack with a varying degree of slippage with respect to neighbouring stacks, altering the close contacts between them, and represent two potential alternative homostructural arrangements for the parent compound. Structure redeterminations of the bromosalicylic acids 5-BrSalA, 4-BrSalA and 3,5-Br2SalA at 100 K show that the packing efficiency of the cocrystals need not be higher than the parent coformers, based on specific-volume calculations, attributable to the strong O-H...O=C hydrogen bonds of 2.54 Šin the cocrystals.

[Recurrent vulvar rashes in a girl aged 22 months for more than half a year].

A girl, aged 22 months, attended the hospital due to recurrent vulvar rashes for more than half a year. Skin biopsy showed Langerhans cell histiocytosis, and evaluation of systemic conditions showed no systemic involvement. Therefore, the girl was diagnosed with Langerhans cell histiocytosis (skin type). In conclusion, for rashes on the vulva alone, if there are no specific clinical manifestations, the possibility of Langerhans cell histiocytosis should be considered after molluscum contagiosum, sexually transmitted diseases, and Fordyce disease are excluded.

Stability and NMR Chemical Shift of Amorphous Precursors of Methane Hydrate: Insights from Dispersion-Corrected Density Functional Theory Calculations Combined with Machine Learning.

Clathrate hydrates of natural gases are important backup energy sources. It is thus of great significance to explore the nucleation process of hydrates. Hydrate clusters are building blocks of crystalline hydrates and represent the initial stage of hydrate nucleation. Using dispersion-corrected density functional theory (DFT-D) combined with machine learning, herein, we systematically investigate the evolution of stabilities and nuclear magnetic resonance (NMR) chemical shifts of amorphous precursors from monocage clusters CH4(H2O)n (n = 16-24) to decacage clusters (CH4)10(H2O)n (n = 121-125). Compared with planelike configurations, the close-packed structures formed by the water-cage clusters are energetically favorable. The 512 cages are dominant, and the emerging amorphous precursors may be part of sII hydrates at the initial stage of nucleation. Based on our data set, the possible initial fusion pathways for water-cage clusters are proposed. In addition, the 13C NMR chemical shifts for encapsulated methane molecules also showed regular changes during the fusion of water-cage clusters. Machine learning can reproduce the DFT-D results well, providing a structure-energy-property landscape that could be used to predict the energy and NMR chemical shifts of such multicages with more water molecules. These theoretical results present vital insights into the hydrate nucleation from a unique perspective.

A Photonic crystal fiber with large effective refractive index separation and low dispersion.

A photonic crystal fiber (PCF) structure with a ring-core and 5 well-ordered semiellipse air-holes has been creatively proposed. Through a comparison between the structures with a high refractive index (RI) ring-core and the structure without, it conclude that a PCF with a high RI ring-core can work better. Schott SF57 was elected as the substrate material of ring-core. This paper compares the effects of long-axis and short-axis changes on the PCF and selects the optimal solution. Especially TE0,1 mode's dispersion is maintained between 0 and 3 ps / (nm · km) ranging from 1.45 µm to 1.65 µm. This property can be used to generate a supercontinuum with 200 µm long zero dispersion wavelength (ZDM). In addition, Δneff reaches up to 10-3, which enables the near -degeneracy of the eigenmodes to be almost neglected. The proposed PCF structure will have great application value in the field of optical communications.

[Recurrent skin blisters for more than 7 months in a girl aged 15 months].

A girl, aged 15 months, attended the hospital due to recurrent skin erythema, blisters, and desquamation for more than 7 months. Giemsa staining and immunohistochemical staining showed mast cell infiltration and degranulation. Hematoxylin staining showed spinous layer edema and blister formation under the epidermis, with a large amount of serous fluid and a small number of inflammatory cells in the blister. Marked edema was observed in the dermis, with diffused mononuclear cell infiltration. The girl was diagnosed with mastocytosis. Mastocytosis should be considered for children with recurrent skin erythema and blisters.

Theoretical Studies on the Fluorescence Enhancement of Benzaldehydes by Intermolecular Hydrogen Bonding.

Density functional theory and its time-dependent extension are employed to investigate the intermolecular hydrogen-bonding- (Inter-HB-) induced fluorescence enhancement of benzaldehydes. The fluorescence quenching mechanism of benzaldehydes in chloroform is attributed to the low energy gap between the lowest ππ* and nπ* states at Franck-Condon (FC) point and conical interaction between ππ* and nπ* states. The Inter-HB between the aldehyde group of benzaldehydes and methanol can considerably increase the energy gap between ππ* and nπ* states at the FC point, and this type of hydrogen bond is strengthened in the excited ππ* state, thereby precluding the nonradiative transition from nπ* state and enhancing fluorescence. The Inter-HB between the hydroxy group of benzaldehydes and methanol is considerably stronger than that between the aldehyde group of benzaldehydes and methanol; which has a little effect on the energy gap of ππ* and nπ* states. Therefore, if Inter-HB exists between the hydroxy group of benzaldehydes and methanol, then it will play an important role in ground and excited ππ* states, and fluorescence enhancement by Inter-HB between the aldehyde group of benzaldehydes and methanol will be absent. Our proposed mechanism can explain the experimental observations and differs from that of "proximity effects".

Cage fusion from bi-cages to tri-cages during nucleation of methane hydrate: a DFT-D simulation.

Water-cage clusters encapsulating guest molecules are the basic components of hydrate crystal structures. Herein, we investigated the fusion process from bi-cages to tri-cages to probe the nucleation mechanism at the initial stage of CH4 hydrate formation by employing dispersion-corrected density functional theory. We found that tri-cages possess high stability by sharing three, rather than two, polygonal faces. In addition, any mono-cage combined with a nonstandard 4151062 cage could achieve considerable stability regardless of which face is shared; this finding illustrates that 4151062 cages are more likely to appear at the early stages of CH4 hydrate nucleation than other nonstandard cages. We then simulated the Raman spectra of CH4 molecules in water-cage to characterize the spectral characteristics of the CH4 hydrate. The C-H symmetric stretching frequency of encapsulated CH4 molecules red-shifted with increasing mono-cage size, which follows the prediction of the "loose cage-tight cage" model. The symmetric stretching vibrational frequencies of trapped CH4 molecules in the tri-cage revealed a clear red-shift compared with those in the component mono- and bi-cages. The cage fusion process and spectroscopic properties described in this work are expected to provide new atomistic insights into CH4 hydrates at the initial nucleation stage.