In Vivo Validation of an In Situ Calibration Bead as a Reference for Backscatter Coefficient Calculation.

OBJECTIVE: The study described here was aimed at assessing the capability of quantitative ultrasound (QUS) based on the backscatter coefficient (BSC) for classifying disease states, such as breast cancer response to neoadjuvant chemotherapy and quantification of fatty liver disease. We evaluated the effectiveness of an in situ titanium (Ti) bead as a reference target in calibrating the system and mitigating attenuation and transmission loss effects on BSC estimation. METHODS: Traditional BSC estimation methods require external references for calibration, which do not account for ultrasound attenuation or transmission losses through tissues. To address this issue, we used an in situ Ti bead as a reference target, because it can be used to calibrate the system and mitigate the attenuation and transmission loss effects on estimation of the BSC. The capabilities of the in situ calibration approach were assessed by quantifying consistency of BSC estimates from rabbit mammary tumors (N = 21). Specifically, mammary tumors were grown in rabbits and when a tumor reached ≥1 cm in size, a 2 mm Ti bead was implanted in the tumor as a radiological marker and a calibration source for ultrasound. Three days later, the tumors were scanned with an L-14/5 38 array transducer connected to a SonixOne scanner with and without a slab of pork belly placed on top of the tumors. The pork belly acted as an additional source of attenuation and transmission loss. QUS parameters, specifically effective scatterer diameter (ESD) and effective acoustic concentration (EAC), were calculated using calibration spectra from both an external reference phantom and the Ti bead. RESULTS: For ESD estimation, the 95% confidence interval between measurements with and without the pork belly layer was 6.0, 27.4 using the in situ bead and 114, 135.1 with the external reference phantom. For EAC estimation, the 95% confidence intervals were -8.1, 0.5 for the bead and -41.5, -32.2 for the phantom. These results indicate that the in situ bead method has reduced bias in QUS estimates because of intervening tissue losses. CONCLUSION: The use of an in situ Ti bead as a radiological marker not only serves its traditional role but also effectively acts as a calibration target for QUS methods. This approach accounts for attenuation and transmission losses in tissue, resulting in more accurate QUS estimates and offering a promising method for enhanced disease state classification in clinical settings.

Spectral-based Quantitative Ultrasound Imaging Processing Techniques: Comparisons of RF Versus IQ Approaches.

Quantitative ultrasound (QUS) is an imaging technique which includes spectral-based parameterization. Typical spectral-based parameters include the backscatter coefficient (BSC) and attenuation coefficient slope (ACS). Traditionally, spectral-based QUS relies on the radio frequency (RF) signal to calculate the spectral-based parameters. Many clinical and research scanners only provide the in-phase and quadrature (IQ) signal. To acquire the RF data, the common approach is to convert IQ signal back into RF signal via mixing with a carrier frequency. In this study, we hypothesize that the performance, that is, accuracy and precision, of spectral-based parameters calculated directly from IQ data is as good as or better than using converted RF data. To test this hypothesis, estimation of the BSC and ACS using RF and IQ data from software, physical phantoms and in vivo rabbit data were analyzed and compared. The results indicated that there were only small differences in estimates of the BSC between when using the original RF, the IQ derived from the original RF and the RF reconverted from the IQ, that is, root mean square errors (RMSEs) were less than 0.04. Furthermore, the structural similarity index measure (SSIM) was calculated for ACS maps with a value greater than 0.96 for maps created using the original RF, IQ data and reconverted RF. On the other hand, the processing time using the IQ data compared to RF data were substantially less, that is, reduced by more than a factor of two. Therefore, this study confirms two things: (1) there is no need to convert IQ data back to RF data for conducting spectral-based QUS analysis, because the conversion from IQ back into RF data can introduce artifacts. (2) For the implementation of real-time QUS, there is an advantage to convert the original RF data into IQ data to conduct spectral-based QUS analysis because IQ data-based QUS can improve processing speed.

In vivo validation of an in situ calibration bead as a reference for backscatter coefficient calculation.

Objectives: The study aims to assess the capability of Quantitative Ultrasound (QUS) based on the backscatter coefficient (BSC) for classifying disease states, such as breast cancer response to neoadjuvant chemotherapy and quantifying fatty liver disease. We evaluate the effectiveness of an in situ titanium (Ti) bead as a reference target in calibrating the system and mitigating attenuation and transmission loss effects on BSC estimation. Methods: Traditional BSC estimation methods require external references for calibration, which do not account for ultrasound attenuation or transmission losses through tissues. To address this issue, we use an in situ titanium (Ti) bead as a reference target, because it can be used to calibrate the system and mitigate the attenuation and transmission loss effects on estimation of the BSC. The capabilities of the in situ calibration approach were assessed by quantifying consistency of BSC estimates from rabbit mammary tumors (N=21). Specifically, mammary tumors were grown in rabbits and when a tumor reached 1 cm or greater in size, a 2-mm Ti bead was implanted into the tumor as a radiological marker and a calibration source for ultrasound. Three days later, the tumors were scanned with a L-14/5 38 array transducer connected to a SonixOne scanner with and without a slab of pork belly placed on top of the tumors. The pork belly acted as an additional source of attenuation and transmission loss. QUS parameters, specifically effective scatterer diameter (ESD) and effective acoustic concentration (EAC), were calculated using calibration spectra from both an external reference phantom and the Ti bead. Results: For ESD estimation, the 95% confidence interval between measurements with and without the pork belly layer was (6.0,27.4) using the in situ bead and (114, 135.1) with the external reference phantom. For EAC estimation, the 95% confidence interval were (-8.1, 0.5) for the bead and (-41.5, -32.2) for the phantom. These results indicate that the in situ bead method shows reduced bias in QUS estimates due to intervening tissue losses. Conclusions: The use of an in situ Ti bead as a radiological marker not only serves its traditional role but also effectively acts as a calibration target for QUS methods. This approach accounts for attenuation and transmission losses in tissue, resulting in more accurate QUS estimates and offering a promising method for enhanced disease state classification in clinical settings.

DNA Repair-Responsive Engineered Whole Cell Microbial Sensors for Sensitive and High-Throughput Screening of Genotoxic Impurities.

Genotoxic impurities (GTIs) occurred in drugs, and food and environment pose a threat to human health. Accurate and sensitive evaluation of GTIs is of significance. Ames assay is the existing gold standard method. However, the pathogenic bacteria model lacks metabolic enzymes and requires mass GTIs, leading to insufficient safety, accuracy, and sensitivity. Whole-cell microbial sensors (WCMSs) can use normal strains to simulate the metabolic environment, achieving safe, sensitive, and high-throughput detection and evaluation for GTIs. Here, based on whether GTIs causing DNA alkylation required metabolic enzymes or not, two DNA repair-responsive engineered WCMS systems were constructed including Escherichia coli-WCMS and yeast-WCMS. A DNA repair-responsive promoter as a sensing element was coupled with an enhanced green fluorescent protein as a reporter to construct plasmids for introduction into WCMS. The ada promoter was screened out in the E. coli-WCMS, while the MAG1 promoter was selected for the yeast-WCMS. Different E. coli and yeast strains were modified by gene knockout and mutation to eliminate the interference and enhance the GTI retention in cells and further improved the sensitivity. Finally, GTI consumption of WCMS for the evaluation of methyl methanesulfonate (MMS) and nitrosamines was decreased to 0.46-8.53 µg and 0.068 ng-2.65 µg, respectively, decreasing 2-3 orders of magnitude compared to traditional methods. This study provided a novel approach to measure GTIs with different DNA damage pathways at a molecular level and facilitated the high-throughput screening and sensitive evaluation of GTIs.

The Comprehensive Effects of Carassius auratus Complex Formula against Lipid Accumulation, Hepatocarcinogenesis, and COVID-19 Pathogenesis via Stabilized G-Quadruplex and Reduced Cell Senescence.

Carassius auratus complex formula (CACF) is a traditional Chinese medicine known for its antidiabetic effects. Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide, and there are currently no effective therapies for advanced HCC. This study explores the comprehensive effects and possible mechanisms of CACF on HCC. The results show that CACF reduces the viability of hepatoma cells in vitro, while benefiting normal hepatocytes. In addition, CACF inhibits hepatoma cell growth in a zebrafish xenotransplantation model and decreases lipid accumulation, represses inflammation and cell proliferation markers in fatty acid translocase (CD36) transgenic zebrafish, and inhibits the expression of cell proliferation and ß-catenin downstream targets in telomerase (tert) transgenic zebrafish models. Ingenuity Pathway Analysis reveals that CACF exerts multiple functions, including reduction of inflammation and inhibition of lipid transporter and PPAR signaling pathway. Surprisingly, CACF also regulates the expression of genes and reduces coronavirus infection and pathogenesis in a zebrafish model. CACF treatment is validated to regulate the expression of genes for anti-coronavirus activity. Mechanistically, CACF stabilizes G-quadruplex and reduces cell senescence associated ß-galactosidase activity. In summary, CACF may be a promising therapeutic agent with multiple functions including anticancer, anti-inflammation, and anti-microorganisms in a zebrafish model.

Dual signal amplified electrochemical aptasensor based on PEI-functionalized GO and ROP for highly sensitive detection of cTnI.

Cardiac troponin I (cTnI) is considered as the gold standard for the diagnosis of acute myocardial infarction (AMI) because of its excellent specificity and sensitivity. Herein, a novel aptasensor based on the dual signal amplification strategy of Polyethyleneimine functionalized Graphene oxide (GO) and ring-opening polymerization (ROP) for the first time was successfully constructed to achieve high sensitivity detection of cTnI. Briefly, cTnI-aptamer 1 (Apt1) was immobilized on the surface of gold electrode by self-assembly of Au-S bonds to specifically capture cTnI. After specific recognition of cTnI, Apt2 coated PEI-functionalized GO composites acted as macroinitiators for the subsequent ROP reaction. Next, α-amino acid-N-carboxylic acid anhydride ferrocene derivatives (NCA-Fc), the monomer for ROP reaction, was added to the electrode surface. The combined application of PEI-functionalized GO and NCA-Fc better achieves the high sensitivity and signal amplification of the aptasensor. Under optimal conditions, the aptasensor exhibited a wide linear range of 10 fg mL-1 to 10 ng mL-1 and the limit of detection was 3.78 fg mL-1. Moreover, this method displayed the advantages of good selectivity, simple operation and excellent stability. Meanwhile, the aptasensor had good accuracy and applicability even in real serum samples analysis, demonstrating its considerable application potential in biomedical assays.

Breast Milk-Derived Limosilactobacillus reuteri Prevents Atopic Dermatitis in Mice via Activating Retinol Absorption and Metabolism in Peyer's Patches.

SCOPE: Supplementing Limosilactobacillus reuteri Fn041, a breast milk-derived probiotic from agricultural and pastoral areas, to maternal mice during late pregnancy and lactation prevents atopic dermatitis (AD) in offspring. This study aims to elucidate the molecular mechanism of Fn041-mediated immune regulation. METHODS AND RESULTS: Fn041 is administered prenatal and postnatal to maternal mice, and to offspring after weaning. The ears are administered with calcipotriol to induce AD. Fn041 treatment significantly alleviates ear inflammation, and reduces mast cell infiltration. Fn041 treatment upregulates and downregulates intestinal ZO-1 and Claudin-2 mRNA expression, respectively. Transcriptome analysis of Peyer's patches reveals that pathways related to DNA damage repair are activated in AD mice, which is inhibited by Fn041 treatment. Fn041 activates pathways related to retinol absorption and metabolism. Untargeted metabolomic analysis reveals that Fn041 treatment increases plasma retinol and kynurenine. Fn041 treatment does not significantly alter the overall cecal microbiota profile, only increases the relative abundances of Ligilactobacillus apodemi, Ligilactobacillus murinus, Akkermansia muciniphila, and Bacteroides thetaiotaomicron. CONCLUSIONS: Fn041 induces anti-AD immune responses directly by promoting the absorption and metabolism of retinol in Peyer's patches, and plays an indirect role by strengthening the mucosal barrier and increasing the abundance of specific anti-AD bacteria in the cecum.

Identifying and overcoming limitations with in situ calibration beads for quantitative ultrasound.

Ensuring the consistency of spectral-based quantitative ultrasound estimates in vivo necessitates accounting for diffraction, system effects, and propagation losses encountered in the tissue. Accounting for diffraction and system effects is typically achieved through planar reflector or reference phantom methods; however, neither of these is able to account for the tissue losses present in vivo between the ultrasound probe and the region of interest. In previous work, the feasibility of small titanium beads as in situ calibration targets (0.5-2 mm in diameter) was investigated. In this study, the importance of bead size for the calibration signal, the role of multiple echoes coming from the calibration bead, and sampling of the bead signal laterally through beam translation were examined. This work demonstrates that although the titanium beads naturally produce multiple reverberant echoes, time-windowing of the first echo provides the smoothest calibration spectrum for backscatter coefficient calculation. When translating the beam across the bead, the amplitude of the echo decreases rapidly as the beam moves across and past the bead. Therefore, to obtain consistent calibration signals from the bead, lateral interpolation is needed to approximate signals coming from the center of the bead with respect to the beam.

Prevention of High-Fat Diet-Induced Hypercholesterolemia by Lactobacillus reuteri Fn041 Through Promoting Cholesterol and Bile Salt Excretion and Intestinal Mucosal Barrier Functions.

Objectives: Lactobacillus reuteri Fn041 (Fn041) is a probiotic isolated from immunoglobulin A coated microbiota in the human breast milk of Gannan in China with a low incidence of hypercholesterolemia. This study aims to explore the role and mechanism of Fn041 in preventing hypercholesterolemia caused by a high-fat diet in mice. Methods: C57BL/6N mice were fed a low-fat diet or a high-fat diet and gavage with Fn041 and Lactobacillus rhamnosus GG (LGG) for 8 weeks. Results: Both Fn041 and LGG prevented the occurrence of hypercholesterolemia, liver and testicular fat accumulation. In addition, a high-fat diet causes intestinal dysbiosis and mucosal barrier damage, which is associated with hypercholesterolemia. Fn041 prevented the high-fat diet-induced reduction in alpha diversity of intestinal microbiota and intestinal mucosal barrier damage. Fn041 treatment significantly increased fecal total cholesterol and total bile acids. Conclusions: Fn041 prevented hypercholesterolemia by enhancing cholesterol excretion and mucosal barrier function.

Selenium-containing tea polysaccharides ameliorate DSS-induced ulcerative colitis via enhancing the intestinal barrier and regulating the gut microbiota.

Both tea polysaccharides and selenium have certain remission potential for ulcerative colitis (UC), but few reports focused on natural selenium-containing tea polysaccharides. The purpose of this study was to isolate a selenium-containing tea polysaccharide (ASeTP) and determine its structure and effects on UC. Results showed that ASeTP was primarily composed of three purified, ß-pyranoside-linked, protein-binding polysaccharides (SeTP-1, SeTP-2, and SeTP-3) with SeOC, OSeO, and SeO linkages. Specifically, SeTP-1 was a neutral heteropolysaccharide principally composed of mannose, glucose, galactose, xylose, and arabinose, while SeTP-2 and SeTP-3 were acidic heteropolysaccharides due to the existence of glucuronic acid. ASeTP effectively alleviated the symptoms of weight loss, colon shortens, and disease activity index scores increase in dextran sodium sulfate (DSS)-induced colitis mice. ASeTP attenuated the histological damage and maintained the colonic mucosal barrier via up-regulating the expression of occludin, claudin-1, and zona occludens-1 (ZO-1). ASeTP suppressed the levels of pro-inflammatory cytokines and enhanced the antioxidant capacity of colon tissue. Besides, ASeTP beneficially increased the selenium content of the colon. Furthermore, ASeTP remodeled the gut microbiota by accelerating the proliferation of beneficial bacteria and inhibiting pathogenic microorganisms. Thus, ASeTP has the potential to be a functional food against colitis.

Purification, characterization and antioxidant activity of selenium-containing polysaccharides from pennycress (Thlaspi arvense L.).

Selenium polysaccharides have antioxidant, anti-tumor and other activities. Tea selenium polysaccharides and Lentinan selenium polysaccharides, etc. have been studied. Pennycress is a super-rich selenium plant and there are few studies about pennycress selenium-containing polysaccharides. In this study, the following researches were carried out on pennycress selenium polysaccharides: Selenium-containing polysaccharides were extracted from the leaves of pennycress (Thlaspi arvense L.) using an efficient subcritical water extraction (SWE) process. After purification, two fractions (Se-PPS1 and Se-PPS3) were obtained and subjected to structural identification. The results showed that the molecular weights of Se-PPS1 and Se-PPS3 were 4.2 × 104 Da and 4.5 × 104 Da, respectively. Se-PPS1 is mainly comprised of glucose, galactose, and xylose. Se-PPS3 consists of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, and arabinose. Fourier transform infrared spectroscopy (FT-IR) analysis showed that the two fractions had absorption spectra typical of selenium esters and spectral characteristics of polysaccharides. The glycosidic linkages were determined by Gas chromatography-mass spectrometry (GC-MS) and Nuclear magnetic resonance (NMR). The two fractions have (1 â†’ 6)-ß-D- Galp and T-α-D- Glcp configurations. Moreover, in vitro antioxidant activity assays showed that Se-PPS1 and Se-PPS3 exhibited effective radical-scavenging abilities, suggesting they have potential applications as natural antioxidants in food and medicine.

Prevention of Atopic Dermatitis in Mice by Lactobacillus Reuteri Fn041 Through Induction of Regulatory T Cells and Modulation of the Gut Microbiota.

SCOPE: The development of atopic dermatitis (AD) in infants is closely related to the lagging development of intestinal microbiota, including that inoculated by breast milk bacteria, and immune development. Lactobacillus reuteri Fn041 is a secretory immunoglobulin A (sIgA) -coated bacterium derived from human milk. METHODS AND RESULTS: We intervene with L. reuteri Fn041 in maternal and offspring BALB/C mice during late gestation and lactation and after weaning of the pups, respectively. AD is then induced with MC903. L. reuteri Fn041 significantly suppresses AD symptoms such as skin swelling, mast cell, and eosinophil infiltration. This effect is attributed to the regulation of the systemic Th1 and Th2 cytokine ratios and the promotion of CD4+ CD25+ Foxp3+ regulatory T cell proliferation in mesenteric lymph nodes. It is also associated with the regulation of intestinal microbiota, particularly promoting Lactobacillus and Akkermansia. CONCLUSION: Our study strengthens the understanding that breast milk-derived sIgA coated potential probiotics are involved in the development of infant intestinal microbiota, thus promoting immune development and preventing allergic diseases, and expanding the knowledge of breast milk sIgA and bacterial interactions on infant immune development.

Depletion of gut secretory immunoglobulin A coated Lactobacillus reuteri is associated with gestational diabetes mellitus-related intestinal mucosal barrier damage.

Changes in secretory immunoglobulin A (SIgA) coated bacteria from early to late pregnancy were associated with the development of gestational diabetes mellitus (GDM). SIgA coated beneficial gut bacteria, which are depleted in GDM, are potential probiotics for the prevention of GDM. We investigated blood biochemistry, chronic inflammation, mucosal barrier biomarkers and faecal SIgA coated microbiota in healthy early pregnancy (T1H, n = 50), late pregnancy (T3H, n = 30) and women with GDM (T3D, n = 27). The "leaky gut" markers, zonulin and lipopolysaccharide (LPS), significantly increased in T3D compared to the T3H group. The Shannon index of SIgA coated microbiota was elevated in late pregnancy compared to early pregnancy and was the highest in the T3D group (p < 0.001). The T3D group was enriched in SIgA coated Escherichia and Streptococcus and depleted in Lactobacillus and Bifidobacterium. Blood glucose (BG) positively correlated with zonulin (p < 0.001) and LPS (p < 0.05). Lactobacillus reuteri negatively correlated with BG (p < 0.05), zonulin (p < 0.05) and LPS (p < 0.01). Lactobacillus reuteri QS01 isolated from the feces of T1H significantly reduced LPS released by the gut microbiota of GDM individuals in vitro. In conclusion, GDM may be related to intestinal mucosal damage and inflammation-induced dysbiosis of SIgA coated microbiota. SIgA coated L. reuteri can reduce the level of LPS of GDM in vitro.

[Antiprotozoal Activities of Eucalyptus Extracts and Their Chemical Components].

Eucalyptus is a fast-growing plant with rich activities. The roots, stems and leaves of Eucalyptus all have medicinal values. Extracts from different parts of various kinds of Eucalyptus show antiparasitic effects, not only the repelling and killing effects on ectoparasites such as ticks and mites, but also the antiparasitic activities against endoparasites such as helminth and protozoa. This paper reviews the effects of Eucalyptus extracts and their chemical componets on protozoa including flagellates, sporozoan and ciliates.

[In vitro Killing Effect of Eucalyptus robusta Leaves Extract on Echinococcus granulosus Protoscolices].

Objective: To investigate the effect of Eucalyptus robusta leaves extract against Echinococcus granulosus protoscolices in vitro. Methods: Mature leaves of Eucalyptus robusta were collected on 24th day in each month from January to December 2012, and air-dried in the room. Ultrasonic extraction of the leaves was done with 4 solvents with different polarity, petroleum ether, dichloromethane, ethyl acetate and anhydrous ethanol. Protoscolices were incubated with the extract at various concentrations for 72 h, and mortality and median lethal dose（LC50） was calculated. Results: The extracts were different in characteristics and yield. The petroleum ether extract was in the form of black oil, while dichloromethane, ethyl acetate and anhydrous ethanol extracts were in the form of dark green, pink and white powder respectively. The average yields by petroleum ether, dichloromethane, ethyl acetate, and anhydrous ethanol were 4.4%, 2.1%, 2.3% and 2.3%, respectively. The extract yield was highest for petroleum ether, with a yield of 5.4% in May. The mortality of protoscoleces in all monthly groups of petroleum ether and dichloromethane extracts reached 100% with the concentration of 100 µg/ml and the same mortality reached in most groups of petroleum ether extracts with the concentration of 50 µg/ml. The effects of dichloromethane extracts were less than petroleum ether extracts, but significantly stronger than those of ethyl acetate and ehanol extracts. Further studies conducted on petroleum ether and dichloromethane extracts showed, the lethal effect of petroleum ether extract ranked in month of preparation from strong to weak as June>March>November>April>February>May>October>August>December>July>January>September. In June, the LC50 was 2.577 µg/ml and 95% confidence interval was 0.85-6.22 µg/ml. The lethal effect of dichlorom ethane extract ranked in month of preparation from strong to weak as November>May>October>April>July>December>June>September>August>February>March>January. In November, the LC50 was 21.85 µg/ml, and 95% confidence interval was 12.38-36.28 µg/ml. Conclusion: The Eucalyptus robusta leaves contain potential compounds against Echinococcus granulosus. Further experiments of isolation, analysis and identification are needed.

Intracellular cleavable poly(2-dimethylaminoethyl methacrylate) functionalized mesoporous silica nanoparticles for efficient siRNA delivery in vitro and in vivo.

A low cytotoxicity and high efficiency delivery system with the advantages of low cost and facile fabrication is needed for the application of small interfering RNA (siRNA) delivery both in vitro and in vivo. For these prerequisites, cationic polymer-mesoporous silica nanoparticles (ssCP-MSNs) were prepared by surface functionalized mesoporous silica nanoparticles with disulfide bond cross-linked poly(2-dimethylaminoethyl methacrylate) (PDMAEMA). In vitro and in vivo evaluations were performed. The synthesized ssCP-MSNs are 100-150 nm in diameter with a pore size of 10 nm and a positively charged surface with a high zeta potential of 27 mV. Consequently, the ssCP-MSNs showed an excellent binding capacity for siRNA, and an enhancement in the cell uptake and cytosolic availability of siRNA. Furthermore, the intracellular reducing cleavage of the disulfide bonds cross-linking the PDMAEMA segments led to intracellular cleavage of PDMAEMA from ssCP-MSNs, which facilitated the intracellular triggered release of siRNA. Therefore, promoted RNA interference was observed in HeLa-Luc cells, which was equal to that of Lipofectamine 2000. Significantly, compared to Lipofectamine 2000, the ssCP-MSNs were more biocompatible, with low cytotoxicity (even non-cytotoxicity) and promotion of cell proliferation to HeLa-Luc cells. The in vivo systemic distribution studies certified that ssCP-MSNs/siRNA could prolong the duration of siRNA in vivo, and that they accumulated in the adrenal gland, liver, lung, spleen, kidney, heart and thymus after intravenous injection. Encouragingly, with the ability to deliver siRNA to a tumor, ssCP-MSNs/siRNA showed a tumor suppression effect in the HeLa-Luc xenograft murine model after intravenous injection. Therefore, the ssCP-MSNs cationic polymer-mesoporous silica nanoparticles with low cytotoxicity are promising for siRNA delivery.

A modified pseudolikelihood approach for analysis of longitudinal data.

We consider the analysis of longitudinal data when the covariance function is modeled by additional parameters to the mean parameters. In general, inconsistent estimators of the covariance (variance/correlation) parameters will be produced when the "working" correlation matrix is misspecified, which may result in great loss of efficiency of the mean parameter estimators (albeit the consistency is preserved). We consider using different "working" correlation models for the variance and the mean parameters. In particular, we find that an independence working model should be used for estimating the variance parameters to ensure their consistency in case the correlation structure is misspecified. The designated "working" correlation matrices should be used for estimating the mean and the correlation parameters to attain high efficiency for estimating the mean parameters. Simulation studies indicate that the proposed algorithm performs very well. We also applied different estimation procedures to a data set from a clinical trial for illustration.